No colonies with Gibson Assembly. Suggestions? Question 5 answers May 21...(ON at 16°C and inactivation of T4 DNA ligase at 65°C for 10 ...Do you have an idea w...

冬天,让人恋家。家的温馨,在冬日,比任何一个季节都来得亲切踏实。生一堆炉火,泡一杯香茗,或面对宽大的书桌,或半靠软软的沙发,不尽畅然。曾几何时,心底深处默默地期待着冬夜故人的来访, 为友人脱下外套,卸下一身的寒气,说说彼此的境遇,感怀一年来的收获。

在这样一个祥和的冬日,就让我们一起, 收获满满!

为回馈广大用户对NEB的支持与厚爱, 2011年NEB冬季促销活动开始了!


活动一:#R0101-#R0199内切酶、T4DNA连接酶  7折优惠(注: V包装不参加活动)                

活动时间: 2011年11月15日-2012年1月15日


活动二: Phusion超保真DNA聚合酶、OneTaqDNA聚合酶65折优惠
活动时间: 2011年11月15日-2012年2月29日


Science topics: Biological ScienceBioinformaticsBioinformatic ToolsPhylogenetic AnalysisPlasmidsScience topicPlasmids - Science topicPlasmids are extrachromosomal, usually CIRCULAR DNA molecules that are self-replicating and transferable from one organism to another. They are found in a variety of bacterial, archaeal, fungal, algal, and plant species. They are used in GENETIC ENGINEERING as CLONING VECTORS.Questions (3,171)Publications (219,494)Questions related to Plasmids1234567891011… 32

Jan Keiten-Schmitzasked a question related to PlasmidsTwo inserts ligation, three restriction sites - possible in single ligation step?Question2 answersAug 11, 2021Hi,I would like to create a Plasmid from an empty verctor and two Inserts, that are to be fused.The empty vector has HindIII, BamHI and EcoRV sites (in that order)Insert 1 is HindIII-Insert 1-BamHI Insert 2 is BamHI-Insert 2-EcoRVThe end result should be ...-HindIII-Insert 1-BamHI-Insert 2-EcoRV-...(to create a fusion of two proteins in a single ORF).Is this possible in a one-step reaction as described below?I thought of digesting the empty vector with HindIII/EcoRV followed by phosphatase treatment and cleanup. Insert 1 will be digested with HindIII/BamHI Insert 2 will be digested with BamHI/EcoRV.combining the digested (dephosphorylated) vector and both Inserts in a 1:3:3 ratio and ligate in a single stepThanks in advance.JanRelevant answerHanna AlalamAug 11, 2021AnswerThis definitely fine. I have done it multiple times and it works as intended. What matters most here is the competence of your cells,I found most success using high efficiency commercial competent neb-10beta cells. however I would suggest your drop the dephosphorlytion treatment as it is not necessary (you have incompatible ends in your vector) and would just decease the overall all ligation efficiency. View0 Recommendations

Terézia Valkovičováasked a question related to PlasmidsKinase, Ligase and Dpnl in Q5 Site-Directed Mutagenesis (NEB) - What is the principle?Question3 answersAug 11, 2021Hello, I am reading the Q5 Site-Directed Mutagenesis (NEB) protocol. I understand that the phosphorylation step is needed for subsequent ligation. But I wonder what is the principle of removing the template plasmids by Dpnl. Any ideas? Thank you!Relevant answerRahul KumarAug 11, 2021AnswerDpnI will digest the methylated DNA (Your template or Parental plasmid) and what will remain is your amplified mutant plasmid, which is non-methylated, before transformation step so that whatever colonies you will get is from your mutant plasmid and not the parental ones. Most of the common lab bacterial strains are Dam methylation positive which methylate plasmids as well and therefore, for this SDM kit one requirement is to use template plasmid isolated from a Dam positive strain. View3 Recommendations

Sudhakar Pagalasked a question related to PlasmidsWhat is the best and simplest way to create chromosomal integration and fusion of gfp in-frame downstream of a protein-coding gene in E. coli?Question3 answersAug 10, 2021I am trying create a chromosomal integrated gfp reporter fusion in-frame downstream of a gene coding for a toxin in E. coli. I do not want to clone/express this fusion from a plasmid, since I want the fluorescence to be proportional to the toxin expression. In addition, if I express that from a plasmid, use of antibiotic selection to grow them in my downstream experiments will interfere with my conclusions. I am trying to monitor the appearance of persister cells in E. coli. Any suggestion will be highly appreciated.Relevant answerMichael J. BenedikAug 11, 2021AnswerThere are lots of different methods to do what you wish to do. I would probably suggest you look at the Datsenko and Wanner protocol for integrating DNA into the E. coli chromosome. There have been dozens of updates to the protocol and if you review the literature you might find that someone has made something just like what you need. Just search for GFP fusions along with Datsenko and Wanner and see what you find. View0 Recommendations

Keunmin Ken Leeasked a question related to PlasmidsCan plasmid DNA electrophoresis show only linearized band and no supercoiled band?Question7 answersAug 11, 2021Hello.I was looking back at some gel pictures of mine (because downstream work is not working...) and realized the electrophoresis for plasmid DNA is kind of odd.From what I know, when plasmid DNA is run on an agarose gel, there are three bands: circular(or nicked), linear, supercoiled.The picture I have included shows plasmid DNA in the 2nd lane with three bands but the bottom band aligns with single-site restriction digested results (all the other lanes to the right).I ve sent the same plasmid (from the same miniprep) for sequencing and I know the total size of the plasmid is ~4000bp, and this agrees with single restriction results. However, somehow when the plasmid is run on gel without restriction, there seems to be only linearized DNA.So my question is, can there be no supercoiled plasmid after miniprep?Any help would be appreciated.

KakaoTalk_20210811_115755131.jpg3.20 MBRelevant answerMalcolm NobreAug 11, 2021AnswerHelloAfter a miniprep by using a commercial kit, you are supposed to get supercoiled plasmid. At times, you might get three bands namely, supercoiled, linear, and nicked plasmid depending on the kit and the handling procedures.In this case, you could consider the possibility of contamination of the resuspension buffer or the loading buffer with nucleases like endonuclease. Repeated freeze-thaw cycles should also be considered.Best. View1 Recommendation

Jinlong Yuasked a question related to PlasmidsMultigene cloning in plasmid?Question4 answersAug 11, 2021I am planing to express 5 genes (casA-E, originating from E. coli) in S. aureus. In E. coli genome, these genes were arranged as an operon and regulated under one promoter.When I clone these genes into S.aureus plasmid, should I insert the whole operon (casABCDE) under an S.aureus promoter or place each of the genes after five independent promoters (pcasApcasBpcasCpcasDpcasE)? Also, the casABCDE genes in E. coli genome are not in the same reading frame. Some of the genes overlaped with 1~3 nucleotides. If I choose to clone the whole operon, should I clone the operon as a whole or clone casA-E seperately (from ATG to TAA) and ligate them together?MG1655_CRISPR-I-E .dna125.64 KBRelevant answerMichael J. BenedikAug 11, 2021AnswerIt is likely to be fine if you clone the entire operon as one segment downstream of your S.aureus promoter. The arrangement of the genes in the operon being slightly overlapping and in different reading frames is quite normal. View5 Recommendations

Ayelet Avinasked a question related to PlasmidsCan anyone help troubleshooting antibody batch differences?Question8 answersAug 4, 2021Hi everyone,We are producing chimeric antibodies in the lab using HEK293 cells.I m currently struggling with an antibody - which binding activity varies greatly between batches.I use the same plasmid for expression and have tried different expression systems (including EXPI). I ve tried concentrating the antibody using both G and A beads (both worked well).The yield of the antibody is fairly good in any system, but the target binding (assessed by flow cytometry) changes drastically with different batches.Can anyone suggest why this should happen to the same antibody? and how can I troubleshoot this?Much appreciated!AyeletRelevant answerJean-Pierre DangyAug 10, 2021AnswerOK then they should bind very strongly to both type of Sepharose. You could have a look to the Gentle elution solutions kit provided by PIERCE. This has helped me for several difficult antibodiesView0 Recommendations

Irem Celikasked a question related to PlasmidsIs negative strand ORF a problem?Question3 answersAug 3, 2021Hi everyone, I m a student and trying to design a lentivirus plasmid. Planning to insert negative-strand ORFs sequences in vector plasmid. However, if add these ORFs into the plasmid as a negative strand, is it become a problem? If it is, how should I add the ORFs as positive-strand into the vector? Or any idea?Relevant answerVipin BhardwajAug 9, 2021AnswerMake sure you have a promoter to drive its expression on the same side. It only matters if it is not in the line with the promoter. View0 Recommendations

Jiwoo Choiasked a question related to PlasmidsHow can I get high yield DNA in endA+ strain?Question4 answersAug 7, 2021Hello, I am performing cloning using lentivirus and transforming the final clone(11kb) into stbl3. I confirmed that cloning was successful through mini prep. However, I tried to amplify the target DNA with maxi prep, but as shown in the attached image, the target DNA band was too weak. (The measured DNA concentration was 700 ng to 1200 ng/ul !) Also, I think the below band around 200bp, is degraded DNA.I knew that stbl3 has an endA gene, so plasmid DNA can be degraded by endonuclease A when E.coli is lysised. So I did optional wash using AW buffer when mini prep to was endonuclease A. However, there was no endonuclease A wash step in midi and maxi prep. So, I ask a question to get advice on how to increase the yield of target DNA.

그림1.png58.19 KBRelevant answerJiwoo ChoiAug 8, 2021AnswerHi, Narumi Uno ! I used to use Dh5a, but occasionally experienced lentivirus host genome recombination. So I use stbl3 to prevent this. Have a nice day :)View4 Recommendations

Batool Ismailasked a question related to PlasmidsHas anyone worked with Absolute qPCR quantification for bacterial genome?Question6 answersAug 6, 2021Hello there! Does anyone have experience with absolute quantification using SYBR green kit for bacterial dna ? And plasmid as positive control, Could tell me the steps and his advice that i have to do to make the standard curve? thanks in advance Relevant answerJochen WilhelmAug 7, 2021Answer Recommendations

Wesley Hansonasked a question related to PlasmidsWhy is my maxiprep pink?Question7 answersAug 3, 2021I recently conducted a maxiprep of a plasmid of interest. No major issues for the majority of the prep, the only strange thing occurred after the elution when I noticed the color of the final plasmid stock was distinctly pink. Concentration and 260/280 values both looked fine, well within expectations, but the color has me a little concerned. Will this prep still be useful in cell culture transfections? What is likely to have caused this color change? For reference I am using the ZymoPure Maxiprep kit, following the protocol provided and using the vacuum manifold for binding.Relevant answerPriyabrata MeherAug 7, 2021AnswerIt may be due to contaminated culture ...Did you reuse the container(falcon/ conical flask) used for culture ?View0 Recommendations

Paul Carrasked a question related to Plasmids Are there methods for reusability of the same qPCR standard curve plasmid? Question2 answersAug 22, 2018I run a lot of qPCR plates on a regular basis. I end up spending quite a bit of that time preparing multiple serial dilutions of plasmids for each individual plate. If I try to reuse the same serial dilutions multiple times in the same day, my standard curve ends up being more unusable as the day progresses, risking not being able to use the data from a plate and having to re-run it later. Freezing the samples overnight and reusing them the next day doesn t work either. It would save me a lot of time if someone could find a way to stabilize a serial diluted curve that could be continuously reused. Relevant answerPrashant KalvapalleAug 6, 2021AnswerI make my serial dilutions with purified PCR products or gblocks or plasmids in TE buffer spiked in with a blocking agent such as herring sperm DNA (10 ng/ul final conc). I have reused them across 30 freeze thaws over 2 months and have not seen any systematic trend of degradation although the variation is very wide (same Cq - change of 1 log of concentrations). The TE buffer maintains a stable pH to minimize hydrolysis and herring sperm DNA minimizes DNA binding to the plastics over time.The graphs attached show std curve slopes over time, and reconstructed lines with the slopes, and intercepts. The lines span a considerable Cq space, I don t know how it compares to the variability if fresh standards were used each time

BCoV_standard_curve_evolution_yint.png48.70 KBBCoV_standard_curve_evolution_reconstructed.png36.93 KBView0 Recommendations

Xiao Yiasked a question related to PlasmidsWhy DNA polymerase overexpression is toxtic to E. coli?Question5 answersDec 18, 2018I am trying to express T7 DNA polymerase in E. coli on low copy plasmid (pKD46, under pBAD promoter). I observed substantial slowing down of cell growth even without induction.Then I looked into literature to find out that expression of T4, T5, Taq, Pfu DNA polymerases all confer toxicity to E. coli.Provided T7 DNAP has the same 3D fold as E. coli Pol I, where does its toxicity come from?Relevant answerJonathan JonathanAug 6, 2021AnswerHey, it might be too late but can you share the literature that refer to your statement Then I looked into literature to find out that expression of T4, T5, Taq, Pfu DNA polymerases all confer toxicity to E. coli. . thankyou in advance..View0 Recommendations

Jafeth Carrascoasked a question related to PlasmidsHow can I recover more concentration of DNA, Using Easyprep plasmid miniprep kit Bioland Scientific?Question8 answersJul 22, 2021Hello,I am purifying an Addgene plasmid with EasyPrep Plasmid Miniprep Bioland Scientific LLC and I am getting low concentrations (5.0 ng / uL to 29.7 ng / uL). I am eluting 50 uL of Elution buffer (TE buffer, pH8.5), I have used 1.5 to 10 ml of culture. Btw: I want to clone, the enzymatic digestions are not working.Please help me. I m desperate!!!Relevant answerPapri BasakAug 6, 2021AnswerJafeth, then it s clearly column issue. Write to your supplier regarding defective supplyView0 Recommendations

Namra Siddiquiasked a question related to PlasmidsWhy my plasmid is self ligating?Question3 answersAug 5, 2021I am trying to clone a 672 bp gene in pet 28 a vector(BamH1 and XhoI)..I am getting very few colonies,to further confirm I ran the ligated product on agarose gel and found two bands one of plasmid and insert. What is the problem in my ligation .I have tried 4 degree o/n and 25 degree room temperature.Relevant answerPaul RutlandAug 5, 2021AnswerHave you run control samples to show that both enzymes are cutting properly in the buffer that you use.If one enzyme is failing to cut then ligation will be very poor. Also when cutting the insert do you have 3 extra bases outside the enzyme cut site as many enzymes only cut well if there are additional bases.View8 Recommendations

Agnès Rastetterasked a question related to PlasmidsHow do I transfect a large plasmid into PC12 cells with nerve growth factor (NGF) receptor?Question9 answersFeb 26, 2021I have a large 16 kb plasmid, which I need to transfect into PC12 cells. Lipofectamin 2000 didn t work and with GFP alone the transfection rate is very low. I also tried the neon invitrogen transfection system following the article An electroporation protocol for efficient DNA transfection in PC12 cells (Covello G, 2014, Cytotechnology). Result similar to Lipofectamin 2000. Are there people using PC12 cells (CRL-1721, non-adherent and NGF responsive) who might have experience in transfection and who could share some knowledge?Thank you very muchRelevant answerKenny P VuAug 5, 2021AnswerSorry for the much later response, but in my experience with the neon transfection machine, you may need to use different parameters that others have reported on. We used HEK293T cells and the parameters sent by thermo as well as others online didn t work for us, it required slight tweaking. Perhaps some slight differences in cells in different labs even though the same line, I ve seen it happen before for many things. Somethings just don t seem to always carry over, despite coming from a low passage stock. If you haven t been able to get it to work yet I would try an optimization series. The neon machine is mostly used by researchers for siRNA and other small constructs, not plasmids. Are you having trouble with viability as well as efficiency? I found that letting the cells rest for 10-15 min RT after electroporating increased both drastically for my plasmids.16 kb is very large, so you can only really do so much with electroporation. In general the I found that the larger the plasmid the more difficult it tends to be viability wise, but I ve yet to have a problem efficiency wise once optimized. Hopefully you have been able to solve your issue thoughView0 Recommendations

Priya Muruganasked a question related to PlasmidsWhere can I get plasmid cyp121 gene for protein expression ?Question13 answersJul 30, 2021Hi researches, Currently, my research is focusing on discovering new potent drugs to inhibit M. tuberculosis activity to treat globally serious TB disease. Therefore, I m looking for a CYP121 gene for protein expression. Can someone suggest me few ways where I can get the plasmid CYP121 gene (pHAT2/cyp121) and E. coli K12 BL21 (DE3) cell for my protein expression and purification research work, please? Thanks in advance. Relevant answerA researcherAug 3, 2021AnswerPriya Murugan It seems that the plasmid is not commercially available and it is also stored at Addgene. Usually, if you generate a plasmid and publish your work, you should provide other with the plasmid. However, at the moment a lot of people in Europe are on summer vacation, and we are still in the middle of a pandemic. Maybe, you should give Prof. Munro a bit more time and send a nice email again...As an alternative:In the original paperArticle Expression, purification and spectroscopic characterization ... the insert for pHTAB2/Cyp121 was cloned from a cosmid from Stewart Cole from the Pasteur Institute in Paris, France. You might get the cosmid there as well.If none of this works, Prof. Liu from the University of Texas also generated a plasmid for expression and purification of Cyp121. They did not use mycobacterial DNA as a template, but ordered the sequence.Article Crosslinking of Dicyclotyrosine by the Cytochrome P450 Enzym... If even this does not help, you could consider gene synthesis yourself.Kind regards and good luckView5 Recommendations

Edison Loasked a question related to PlasmidsWhat causes my plasmids to be nicked, and will they affect restriction enzyme efficiency?Question3 answersAug 1, 2021Dear all,I am currently preparing a probe for whole-mount in-situ hybridization. I have been given constructs (that are validated by sequencing) and ordered to carry out a transformation. After the transformation, I carried out plasmid kit extraction and realized that most of my plasmids seem to be nicked. Neither did I carry out vortexing during the whole process nor did I allow the P2 (FADP2) Buffer to lyse my cell for too long (I had lysed them for 3 minutes and immediately added the P3 Buffer for neutralization). May I ask whether this is potentially caused by:(1) Using colonies that are not freshly prepared. Mine was prepared two days ago and stored in a 4 degree Celcius fridge.(2) Overgrown of Bacteria Culture. I have been shaking my 4.5ml Broth + 4.5 1000x µl Amplicilin for 20 hours. I know that the optimal time for that is 14~18 hours but I really have no choice at that time.I would also like to ask whether my plasmids being nicked will result in their inability to linearize or it simply will work as to how supercoiled plasmids do? Relevant answerMichael J. BenedikAug 3, 2021AnswerA nicked plasmid will run in a gel with about the same mobility as the line arises plasmid, so running a digested contril alongside is informative. It is also possible that you have supercoiled dimer plasmid . These will all resolve upon digestion. View0 Recommendations

Risabh Sahuasked a question related to PlasmidsProblem in high yield plasmid purification using qiagen midi kit for DENV 2 whole genome containg plasmid. help me?Question2 answersJul 16, 2021I am getting very less concentration of plasmid for Dengue whole genome containing plasmid using qiagen midi kit. concentration is very less ,around 130 ng/ microlitre, which is not good for infection.Relevant answerRisabh SahuAug 2, 2021AnswerThanks Túlio Teruo Yoshinaga . It helped me a lot View0 Recommendations

Shraddha B Jogiasked a question related to PlasmidsUnsuccessful transformation of Rubidium chloride E. coli DH5alpha cells using plasmid DNAQuestion3 answersJul 28, 2021Hello all. So, we transformed RbCl E. coli DH5alpha cells using 50ng of DNA. But, we did not get a single colony. Can anyone explain the reason behind this problem?Note: RbCl comp cells, Ampicillin stock and LB+Amp plates were freshly made.Relevant answerMichael J. BenedikAug 2, 2021AnswerSounds like the issue is the plasmid DNA then. You could try to transform it into some different cells to see if it works, or look at the DNA on a gel to see if you have DNA. View0 Recommendations

Yazen Alnefeesiasked a question related to PlasmidsHow large can the cloned insert be without causing problems in downstream protein expression?Question4 answersAug 2, 2021I want to clone a 4.1kB insert into a 7.2kB plasmid that I plan to transform into BL21. I assume it ll work fine but I m curious what the limits are and why they exist.Relevant answerMichael J. BenedikAug 2, 2021AnswerThere really are not any limits (within reason) but the efficiency of transformation is somewhat reduced as you go up in size. Otherwise it is just a case of ease of manipulation of the clone. Natural plasmids are often hundreds of kb s in size. View4 Recommendations

Mi Nguyen-Tra Leasked a question related to PlasmidsHow can I analyze the coverage and identity between 2 plasmids?Question4 answersJul 30, 2021I have the complete sequences of two plasmids (~200kbp each) and want to compare and calculate the coverage and % identity between them. Could anyone please tell me which tools or softwares can I use? Thank you in advance.Relevant answerMd. Mamunur RashidAug 2, 2021AnswerProvided link could be helpful:Article PlasmidSeeker: Identification of known plasmids from bacteri... View1 Recommendation

Thomas Juanasked a question related to PlasmidsHow to repress expression on a plasmid before integration?Question4 answersApr 17, 2021I would like to know if there is any genetic trick to repress the expression of a reporter on a plasmid but only before integration in the injected cell genome.I m trying to get rid of the expression from the naked plasmid injected in cells, but make it visible only when it is inserted.Thanks for your answer!Relevant answerThomas JuanAug 1, 2021AnswerDear Mohammed, thanks for your answer. But the system you proposed won’t work. Let me rephrase the question maybe: I’m looking for a genetic trick so that if we inject a plasmid in a eucaryote cell, we would have a trancription of a transgene from the genomic integrated plasmid only, and not from episomal copies.View0 Recommendations

Sumaiah Diabasked a question related to PlasmidsFLAG Expression From WT cell?Question3 answersJul 29, 2021HI I am transfecting my DNA into HEK293T cells . The WT cells (which has the plasmid used for cloning) do not show the flag when WB was done , while it was expressing in the mutant i made. Can please someone help with that?Thank youRelevant answerDidier PoncetJul 30, 2021Answeris the Flag fused to something in your WT plasmid? if not only a short protein is make that run out of the gel. does it have a start codon? did somebody used the wt plasmid before? maybe the wt has a mutation that you corrected doing the mutant... did you sequenced the wt and mutant genes? View0 Recommendations

Sumaiah Diabasked a question related to PlasmidsWild type control for transfection?Question2 answersJul 30, 2021Hi AllI have a technical question regarding the transfection control. For the WT control (where we use the parent plasmid for cloning) Should i make a PCR reaction for it , transformation , miniprep and used for transfection ? or use it directly in transfection? I am just starting to learn this technique and i will appreciate any help.Thank youRelevant answerGuy LongepiedJul 30, 2021AnswerYou can amplify a sequence wt and your sequence with the mutation then clone into a expression vector with a tag (flag or myc, Nter or Cter). To check if you are in good conditions for transfection use a vector that express GFP.For your plasmid : do a transformation for amplification, check by PCR your colony, make a miniprepView0 Recommendations

Suchi Chaturvediasked a question related to PlasmidsRegarding western blot troubleshooting ?Question8 answersJul 27, 2021Dear allI am trying to transfect my plasmid of interest in the mcf-7 cells by lipo3000 or calcium phosphate. but after botting observing multiple bands tried every possible condition, including blocking condition variation, increasing antibody dilution, and reducing exposure time, etc. previously I used to observe only the signal band after using this antibody (SC-7392) expected band is at 34 kDa but now observing multiple bands. Now, I am not able to troubleshoot the problem.Please suggest me something to overcome this issue

protein.jpg82.13 KBprotein.jpg82.13 KBRelevant answerEmre BektikJul 29, 2021AnswerI assume you use secondary antibodies, but not conjugated version of primary antibodies. Have you tried using second antibodies raised in different species, e.g. anti-mouse raised in goat? Maybe your secondary is nonspecifically binding, if so, optimizing primary antibody conditions will likely not work. You can also consider shortening the time of second antibody treatment.View5 Recommendations

Mehwish Iftikharasked a question related to PlasmidsWhich position of His-tag is better for protein expression?Question6 answersJul 20, 2021Greetings.I have constructed pet28a plasmid with desired insert and have added His tag at two positions i.e., at C-terminal and N-terminal. However, I only get expression for N-terminal His tagged protein. I have probed optimization conditions for both constructs and only N-tagged terminal has shown expression. I wonder is there any relation of His tag position on protein expression or what could be the reason for it? Thanks.Relevant answerMehwish IftikharJul 29, 2021AnswerSyed Zawar Shah Thank you for the recommendation. View0 Recommendations

Caroline Ferreira de Santanaasked a question related to PlasmidsCould I use TOP10 instead of DH5alpha for plasmid isolation?Question3 answersJul 27, 2021I will perform a transformation in clinical isolates of P. aeruginosa and I need a competent cell to amplification and isolation of my vector pEx18Tc. Relevant answerJohannes StuttmannJul 28, 2021AnswerYes, Top10 is the same as Dh10b (there is a comment on that somewhere in the openwetware Ecoli genotypes), and is perfectly suited for plasmid amplification! We use it for pretty much everything. View4 Recommendations

Suchi Chaturvediasked a question related to PlasmidsLipo 3000 transfectionQuestion4 answersJul 15, 2021Hello allI tried to transect MCF-7 by lipo 3000 using 250 ng, 500ng or 2000ng plasmid DNA for 48 hrs in serum-free media. But unfortunately not able to successfully transfect my plasmid. Secondly, I saw no. Of non-specific bands on my blot from. Here I have several doubts.. 1 is my transfection technique Has issue? 2 or optimization is needed for western blot if yes then what could be the conditions? 3 my primary antibody has issues or it is degraded ( sc-7392)?Lysis was done in 1%NP-40 buffer, 150 mM NaCl, 1 mM EDTA ( 30 min on ice with 30sec vortexing in every 10 min) centrifugation 14000 rpm 4C for 20 min Loaded: 30 ug Blocking : 2 hrs RT 10% BSA in 1x TBST 1 ab: O/N 1:3000 4 C in 1x pbst 2 ab: 2 hrs secondary mouse (1:5000) Relevant answerPankaj GuptaJul 27, 2021AnswerYou did not mention which scale (6/12/24 well plate) you are trying to transfect. Moreover, you must always have positive controls for transfection (GFP/any protein that express). Did you check if your cells or alive after transfection (6-12 hours). Lipo-3000 is toxic in high quantity try to optimize the ratio with DNA. Serum free medium was used only for transfection and serum medium should be added for recovery of cells from stress. Hope this will help.View0 Recommendations

Govindan Raghunathanasked a question related to PlasmidsHow to solve dual plasmid transformation issue in S. cerevisiae? Question4 answersJul 26, 2021I have to transform two plasmids into yeast. One is 8kb with trp marker and other is 14 kb with ura marker. both are 2micron. I used Gietz et al method.First, i tired tansforming both plasmids at same time and after plating on trp/ura plates, no colonies were observed.Secondly, I prepared competent cell containing 8kb plasmid (trp marker) and tired transforming 14 kb plasmid (used 500ng). Still no colonies were observed. But the controls were perfectly alright. i could see lawn of cells in Trp drop out plate (POSITIVE CONTROL). As negative control, cells were plated in ura drop out plate and no colonies were observed. How could I solve this issue?Relevant answerGovindan RaghunathanJul 27, 2021Answeri have no doubt in reagents, i use them routinely for my experiments. I would try transforming only ura plasmid (14 kb) into yeast and see the outcome. thanks Dominique Liger View0 Recommendations

Ashis Kumarasked a question related to Plasmids3D transfection with chitosan hydrogel?Question4 answersJul 22, 2021I am facing a problem with plasmid DNA transfection in the 3D hydrogel system.I am using a two-component hydrogel system, where you mix the Comp A and B at a definite ratio it forms a hydrogel. So what we do is, we mix the mesenchymal stem cells in comp B which is hyaluronic acid, and the plasmid DNA complex prepared using Mirus trans-IT x2 transfection reagent is mixed with comp A which is chitosan. In this way, we make a 3D hydrogel with both MSCs and pDNA complex encapsulated in a 3D scaffold. After encapsulation, it should ideally release the pDNA complex slowly and it should be able to transfect the MSCs in the gel system and produce the GFP signal. Anyone working on the same area can help me understand what would be the reason that I don t see any cells getting transfected with GFP pDNA?Any experts in this field? working with chitosan ?Relevant answerSaurabh MandalJul 26, 2021AnswerWithout a hydrogel system, most of the standard transfection reagents work well with MSC. We have used Lipofectamine 2000, Lipofectamine 3000, ABM DNAfectin, and Polyplus JETprime. These all have worked well for plasmid transfection.View0 Recommendations

Royal Shresthaasked a question related to PlasmidsSDS-Page Gel Question?Question4 answersJul 24, 2021Hello,We’ve run a few SDS page gels in the past few weeks but so far, we have not been getting much bands. We bought a PET-21-b+ plasmid and eventually transformed them. After growing it up in 2xyt and lb, and getting an OD of around 0.7, we followed an SDS page protocol to view Our protein bands. we are using a 12% 30 uL gel and loading about 20 uL of sample into each well.

E160CCAC-C611-4EE1-B752-47CF155832D6.jpeg519.44 KBRelevant answerPaul RutlandJul 26, 2021AnswerIt looks to me like the gel has not set properly. Try freshly made APS and give the gel longer to setView6 Recommendations

Cushla Mckinneyasked a question related to PlasmidsWhy are the SV40PolyA signal and downstream sequence elements inverted in many common expression plasmids?Question2 answersJul 21, 2021Rather than have the SV40 PolyA sequence and 3 elements 5 to 3 on the coding strand, many reporeter expression vectors (such as EGFP) have the PolyA 5 to 3 on the opposite strand (with the end of the 3 element nearest the stop codon and the start of the PolyA furthest away). How does this influence polyadenylation of the reporter mRNA, especially since the sequence will not actually be transcribed?Relevant answerCushla MckinneyJul 26, 2021AnswerThanks Anyu, after a day of trawling through the literature I realised that they represent the early and late polyadenylation SV40 PolyA signals; early and late genes are transcribed off opposite strands. View0 Recommendations

Diptesh Dasasked a question related to PlasmidsNo colonies upon transforming using GENEART site-directed Mutagenesis System?Question5 answersJul 25, 2021I have been trying to perform a single amino acid mutation using a the GENEART site-directed mutagenesis system. I isolated the plasmid, fixed its concentration at 50ng/ml for the mutagenesis PCR (received multiple faint bands on 0.8% agarose) then performed recombination reaction followed by its immediate transformation in already provided chemically competent DH5a cells .Firstly i tried the as per the instructions given in the kit s manual ( no colonies), later I tweaked the transformation protocol ( made fine adjustments such as: increased ice incubation for after transferring recomination reaction by 8 mins increased heat shock duration by 60 sec, etc.) I tried transforming the DH5a cells with the wildtype plasmid and it did give colonies. But no colonies with the recomination reaction whatsoever. Any suggestion might save reactions in the kit for my further experiments. Thanks for your valuable time. Relevant answerDiptesh DasJul 25, 2021AnswerDear Papri, Yes, you are right about the efficiency of the kit, even I found it in several protein engineering publishings. As I am naive to this area of work, I need to standardize it first, however, I can say I am in the process :). As you mention about the digestion of the parental plasmids, I think in this case it happens after paternal plasmids enter the host cell by MrcBC endonuclease. Nevertheless, I shall request you to go through the manual once. I will make good use of your insights. Cheers!!Diptesh geneart_site_directed_mutagenesis_man(2).pdf1.59 MBView0 Recommendations

Naveed Shahzadasked a question related to PlasmidsWhat is the best reagent for the transfection of Hela cells with a large (12kb) plasmid?Question3 answersJul 25, 2021I am trying to transfect Hela cells with a large 12 kb plasmid by using Lipofectamine 2000. I have used various concentrations (1,2,3,4 and 5 ug) of the plasmid and transfection reagents but the maximum efficiency was 30 %. I am not sure if I can get higher efficiency with such a large palsmid?Can someone answer please whether this efficiency is sufficient to check the expression of genes transcribed by the transfected plasmid? Also, help me knowing which reagents work best for the Hela cells?Relevant answerMalcolm NobreJul 25, 2021AnswerHello Naveed Shahzad You could improve on the efficiency.Make sure you have included the points below:1. Transfect cells at 40–80% confluency. Too few cells may cause the culture to grow poorly without cell-to-cell contact and too many cells will result in contact inhibition, making cells resistant to the uptake of foreign DNA.2. Be sure that the passage number is below 40-45.3. Be sure the DNA is free of protein, RNA, chemical and microbial contamination as impurities could decrease the efficiency.I would recommend you to use jetPEI which is a powerful reagent that ensures robust, effective, and reproducible DNA transfection into mammalian cells with low toxicity.Please refer to the link below for more details. Luck.View5 Recommendations

Vikram Kumarasked a question related to PlasmidsDHFR-MTX selection in CHO (DHFR -ve cells ) ?Question3 answersJul 22, 2021Generally CHO (DHFR -ve) cells on transfection with plasmid bearing DHFR gene + Gene of Interest and upon addition of MTX only thoose cells which take up plasmid (containing DHFR + Gene of Interest ) will survive others will die.My doubt 1 is : Generally DHFR is involved in De novo synthesis of Nucleotides, then how the nucleotides are synthesized in CHO (DHFR -ve ) cells?My doubt 2 is : CHO (DHFR -ve ) cells lack DHFR so they couldn t use De novo pathway for nucleotide syntheis but they can use salvage pathway, then after transfection with Plasmid (containing DHFR + Gene of Interest) all the cells will survive due to operation of salvage pathway, now how to distinguish between the transfected cells vs Un transfected cells.I m confused with this DHFR-MTX selection system, could someone please help me to understand this concept, Also please share any referance material.Relevant answerMalcolm NobreJul 25, 2021AnswerVikram Kumar you re welcome.View0 Recommendations

Sumaiah Diabasked a question related to PlasmidsMyc tag antibody ?Question3 answersJul 24, 2021 Hi AllI am recently doing WB fron transfected cells HEK293(using a plasmid tagged by Myc DDK) For protein extraction i used RIP buffer and protease inhibitor.The Myc antibody i sued from Origene (CAT#: TA150014). After probing the blot with the tag i ended up with high background and multiple bands that exist even in the negative control . The protein concentration i started with was 15 mcg and the tag dilution was 1:1000 . I am kind of new to this area and i am reaching out for help. Thank YouRelevant answerAnoop ArunagiriJul 25, 2021AnswerHi,Most Myc tag antibodies I have used so far have the same problem on western. I partially fixed this issue by introducing an overnight blocking step using 5% skimmed milk prepared in 1X TBST. I would encourage you to try this approach, and follow this with three consecutive washes with 1X TBST (5 mins each) before adding the secondary antibody the next day.Good luck!AnoopView5 Recommendations

Alek Ericksonasked a question related to PlasmidsPCR only worked when the tube melted, why?Question3 answersJul 23, 2021Hi, I am linearizing a 8kb lenti puro vector with PCR prior to Gibson assembly. I have precious few reactions in my Gibson kit so I don t want to use something until I m sure what it is. Currently I am preoccupied with the mystery of why the PCR seems to work very well (get expected band) only when I use the tubes that melt in the thermo cycler, but when I run the same reaction with tube strips that never melt, I barely see any band (mostly a smear). Reaction - 51.5uL totalTemplate Plasmid Dna 43ng/uL - 1uLmol. bio. H2O - 35uLDMSO (has been freeze-thawed) - 2.5uL Q5 5X reaction buffer - 10uLQ5 Polymerase - 1uL 10uM Primers (F/R mixed) - 1uLAccording to eurofins genomic and snapgene my primer Tm are 65-66 C. My Program95C - 5min95C - 20 sec61C - 20 sec72C - 3 min25X cycles from steps 2-472C - 5 min 4C - infinityI am currently running an experiment to see if splitting the reaction volume in 2 parts or increasing the Ta to 67C will help. Relevant answerJunjie ShaoJul 23, 2021AnswerHave you checked the Q5 protocol from NEB, the Denaturation is 98oc, not 95oc.View0 Recommendations

Marcin Goławskiasked a question related to PlasmidsHow to increase the length of poly(A) repeat in a plasmid?Question1 answerJul 21, 2021Hello,If one had a plasmid with a 70 bp long sequence of adenines, what would be the best way to increase that length of repeated adenines to 100-130bp? It is not strictly required for the resultant poly(A) to have an exact length but it is preferable.Relevant answerMiaad K. AlkhudhairyJul 23, 2021AnswerThere is no free account, and if you find one, it won t last longView9 Recommendations

Ragan Pitnerasked a question related to PlasmidsWhat s up with my plasmid prep?Question12 answersJul 13, 2021Hi all,I recently ran a restriction digest check of several of my plasmids and one of them was weird. All the plasmids in this picture received the same restriction enzymes and are all loaded at ~3ug per well. I looked at the one in question (second from right) on the nanodrop again and had a beautiful peak with the proper ratios. What do you think is going on here? Excessive salt contamination? It doesn t really look like degradation to me since I still have somewhat distinct bands at the proper locations.Thanks for your help,Ragan

20210712 pAAV Plasmid Gel Analysis_2.jpg76.89 KBRelevant answerAmy KlockoJul 22, 2021AnswerMaybe some salt and/or degradation. A new prep would help with either problem. When you say the first purification yielded the wrong plasmid, what do you mean? Wrong band pattern/sizes? Too much smearing?View5 Recommendations

Marcin Goławskiasked a question related to PlasmidsBest way to asemble and clone into a plasmid a 10kb gene from 1kb fragments?Question5 answersJul 2, 2021If one were to assemble such a large gene from those small fragments into a plasmid, what method would be preferable? Those fragments may be available as a part of a plasmid or as a PCR product.The exact number of fragments isn t explicitly defined here but due to afromentioned constraints, it will be higher than 10, perhaps 12, plus a plasmid backbone.Golden gate method and perhaps Gibson assembly seem to be some possibilities, provided that specialised protocol and products are used.Relevant answerMarcin GoławskiJul 21, 2021AnswerIt d appear now that I can obtain a plasmid containing the desired sequence in a different way but I d like to thank for the answers.View2 Recommendations

Sayanti Halderasked a question related to PlasmidsAnyone help me with size of test plasmid of pET-32 Xa/LIC cloning kit?Question4 answersJul 21, 2021We have transformed the test plasmid of pET-32 Xa/LIC cloning kit with E. coli DH5 alpha competent cells. After 24hours of incubation on LB medium (50 µg/ml ampicillin containing medium) at 37°C, bacterial colony was found. We isolate the test plasmid from the colony by plasmid purification kit and check DNA bands on agarose gel (1%). But we don t know the size of the test plasmid, that s why we are confused whether the transform of test plasmid in E. coli DH5 alpha competent cells was successfully done or DNA bands are because of genomic DNA? (Result of agarose gel is attached)Any comments or suggestions are welcome..!

Test plasmid isolation..jpg1.59 MBRelevant answerGregory DresslerJul 21, 2021AnswerIt is not clear what the size markers are but this looks like a supercoiled plasmid. No I do not think you have the genomic DNA here. Try cutting with a restriction enzyme and see what you get. Your plasmid should be about 5926bp. You can get a map online just by googling the plasmid name. The supercoiled plasmid does not run at the at the size of the linear plasmid, it usually runs at a lower MW. View10 Recommendations

Lakshya Saxenaasked a question related to PlasmidsCan we use agrobact. to clone a plasmid instead of Ecoli?Question4 answersJul 21, 2021While doing CRISPR cas mediated gene knock out in potato, cloning of vector is done in E can we directly clone it in Agrobact. and not transforming it to Ecoli.?Relevant answerJoshua PhilipsJul 21, 2021AnswerHi Lakshya,If you are using a binary plasmid to clone your gene of interest into, you can in theory transform this straight into Agrobacterium without going through E. coli first. I would recommend to go through E. coli for many reasons, e.g., in case you want to confirm the integrity of the clone E. coli will give you better yields in plasmid preps and you will also not co-isolate the disarmed Ti plasmid.View5 Recommendations

Chris Leeasked a question related to PlasmidsIn regards to homologous recombination, will transformed plasmids with fragments that have NOT been integrated into the chromosome affect selection?Question3 answersJul 17, 2021Another way of phrasing this question would be: Is there a chance that a transformed plasmid with homologous sequences does not recombine with the chromosomal DNA? If so, will a cell still be selected due to the plasmid vector containing the selectable marker? Accordingly, if previous statements are true, would one have to run multiple samples through a gel and select the colony without the plasmid (my guess)?Relevant answerMarco PalmaJul 20, 2021AnswerI have constructed several mutants in S. aureus and P. aeruginosa using the allele replacement techniques (double recombination). The plasmids I used had a temperature-sensitive origin of replication, so when you shift the temperature you get rid of the plasmids and conserved the clones with recombined fragments.However, even when you think you had got rid of the plasmid, you need to screen several clones with PCR to identify the good clones because the plasmid in some clones mutate and become resistant to temperature. Even when you don t use this approach of temperature-sensitive plasmid, you need to screen with PCR to identify the good clones that have the recombinant fragment and not the plasmid.View0 Recommendations

Evelyn Garlickasked a question related to PlasmidsTransfection toxicity when plated on glass coverslips?Question5 answersJan 28, 2021I ve been using A549s for the past couple of years to transfect in my protein of interest for imaging. Recently I ve started to experience issues with transfecting cells seeded on glass coverslips. The same plasmid prep, PEI stock, and seeding density results in good transfection efficiency and cell survival in wells where cells are simply seeded on the plastic, but when seeding on glass coverslips almost all the cells in the well round up and die after 24 hours. I ve not previously had this issue, and have tried a couple of different frozen stocks of A549s to ensure it s not a problem with the particular batch of cells themselves. I ve used the same coverslips with HEK293T transfections and not seen this same toxicity, nor do I see A549 cell death if I m not introducing transfection reagents. I m loath to coat the slips with polylysine as this would interfere with some of my downstream applications. If anyone has experienced a similar issue or has any suggestions I d be very grateful! Relevant answerHamadi MadhiJul 20, 2021AnswerLipofectamine 2000 works fine with A549. View0 Recommendations

Aditya Misraasked a question related to PlasmidsCreating an infection reporter plasmid that is transcribed/translated by mammalian cells?Question4 answersJul 18, 2021Hi,I d like to create a plasmid that a pathogen (like Klebsiella pneumoniae) can carry/replicate into the mammalian cells it infects. Then, that plasmid (assuming a small number of them survive being phagocytosed) has a mammalian promoter and downstream of it contains a fluorescent protein that localizes to the nucleus. As a result, a cell that contains KP would fluoresce with a nuclear fluorescent readout. Is this possible? I ve seen engineered KP variants have pET plasmid vectors but I m afraid they have bacterial promoters. How can I have a plasmid vector that contains a mammalian promoter and fluorescent protein but that it ll replicate well in the KP? I ve had an experience where I electroporated some mammalian plasmids into KP but it affected their growth massively. Those plasmids contained a CMV promoter.Relevant answerJohn SchloendornJul 19, 2021AnswerHow would your plasmid get out of the Klebsiella? I think it will stay in the bacterium for as long as it is alive, and it will be destroyed when the Klebsiella is destroyed. But OK we ll assume some of the bacterium makes it into the cytoplasm, and then dies there, and leaks out intact plasmid. It won t actively localize to the nucleus. The best you can hope for is that the cell wants to divide, disassembles its nuclear membrane, and then encloses the plasmid when it reassembles it. To get it to actively go to the nucleus, you would need to do something harebrained, like express a DNA-binding protein containing a mammalian nuclear localization signal that would drag the whole plasmid along. This is not well established. You could do a PhD thesis or 2 on that part alone. So, I hate to rain on your party, but I think none of your scheme will work -- deal with it. But the part that you asked about is actually easy. You design a construct containing all the features you need for mammalian expression, like promoter, Kozak, gene of interest, stop, terminator. Clone it into the pET vector. The mammalian cell will ignore any bacterial expression features on the vector. It does not hurt it to have them there. And expression in the bacterium will not work, for lack of a bacterial ribosome binding site. (just don t put one there...)View5 Recommendations

Niklas Eckert Elfvingasked a question related to PlasmidsDigestion with Nde1 and Spe1 with modified pET24(+) plasmidQuestion2 answersJul 13, 2021Hi,I have been trying simultanious digestion of a modified pET24(+) plasmid with Spe1 and Nde1 in a single mixture. The buffers documentation states 100 % efficiency for Nde1 and 75 % efficiency for Spe1. An agarose gel test I did showed however that Nde1 is not cutting efficiently while Spe1 is cutting fine, and this prevents me from continuing with my biobrick assembly. The modification of the pET24(+) is that an IF1 gene with a Nde1 restriction site is incorporated.I would now like to try to perform the digestion of the plasmid with the restriction enzymes separetly. I am not sure however on how to go about this;Do I need to purify the DNA after the first cut to get rid of the first enzymes buffer before I move on with the next cut? Should I cut with Nde1 or Spe1 first? Any other general tips that you could give is more than welcome!All the best! Relevant answerNiklas Eckert ElfvingJul 16, 2021AnswerThank you for your reply, it was very helpful!I tried a similar automatic protocol tool through Thermo Fisher, as the enzymes were purshased from there, and now the digestion works great! Recommendations

Karen Mentionasked a question related to PlasmidsIs Lipofectamine 3000 sensitive to the expiring date?Question3 answersJul 13, 2021Hi, I used to use lipofectamine 3000 and it worked very well. But recently my same transfections are not working (No DNA editing, while before, the same transfection was giving me 25% editing). I don t know what is the cause. The FACS analysis seems to show expression of the GFP containing plasmids in 20 to 70% of cells.I recently noticed that my lipofectamine 3000 reagents are expired. I used one expired since 2020 and one since April 2021. But none worked.I also noticed my optimem is slightly expired since maybe beginning 2021.Do you know if the lipofectamine 3000 or Optimem are reagents that cannot be used after expiring date (they are both stored in the fridge at +4)Do you have any other idea what can be the problem? I ordered new reagents anyway, so I can compare the transfections once I receive them. But I would like some opinions if people have different ideas.Relevant answerAnindya DuttaJul 15, 2021AnswerExpired media and reagents usage is highly discouraged. I faced a similar problem with my experiment. This makes troubleshooting way more complicated!View0 Recommendations

Samuel Changasked a question related to PlasmidsCan E. coli LB mixture be transfer to a new fresh LB and shake for growing?Question3 answersJul 13, 2021I m now working on a large quantity of DNA cloning which have to repeatedly perform mini-prep and midi-prep. I was been told that some of the plasmids such as pUC57 or pLNCX can first be grow in 3 c.c. LB in polystyrene round bottom tube, half of volume will be use to extract DNA and enzyme check, and if the result was correct, 20 ul of LB bacteria mixture can then be transfer from the rest 1.5 c.c. to a new 100 c.c. LB containing Erlenmeyer flask, than midi-prep.But only for the plasmids size that s about 5000 bps. For a larger plasmid such as pLKO that in our lab is around 9000 bps, this procedure can not be done.That is we must do the mini-prep than re-transform the correct DNA, we can than use it to perform midi-prep.Does someone have theory or experience about it?Relevant answerMichael J. BenedikJul 13, 2021AnswerI can think of no valid reason why size alone would be a factor in your ability to passage a culture as you describe. I think the same protocol would work fine for both size groups. Having said that, if you have a clone(s) that is showing a growth defect, the more times you grow out a culture you increase the likelihood of picking up mutations that alleviate the growth defect. But that is normally more an issue with expressing some proteins and not so much a size issue. It might also be there is trait with the pLKO plasmid that might be selected against, but again this is not a size issue but rather a plasmid genotype/phenotype issue. View15 Recommendations

Dharmender Guptaasked a question related to PlasmidsHow many plasmid can transform in single bacterial cells and how to perform these type of transformation?Question4 answersJul 9, 2021How to remove natural plasmids present in bacterial cells in a manner so that we can use that cells for our research?Relevant answerAndrew JenkinsJul 12, 2021AnswerThere is, as far as I know, no completely reliable method for curing natural plasmids. However, there are several conditions that favour loss of plasmids, such as growing the cells in the presence of acridine orange or at sublethal temperatures. There are also some bacteriophages that have conjugative pili as their binding site, and these might be useful as selective agents if your plasmids encode the right kind of pili. On the other hand, why bother? As long as the resident plasmids belong to a different incompatibility group from the plasmids you want to use for genetic manipulation, they are not a barrier to transformation unless they encode restriction enzymes (some do), though, of course, they will complicate molecular analysis. As regards transformation, there is no theoretical limit to how many plasmids could be transformed at once, but the process is so infrequent that the chance of one cell being transformed by more than one plasmid is tiny. In addition, since the transforming material usually belongs to a single incompatibility group, the plasmids would not be stably maintained. Note that transforming natural bacterial strains is a great deal more difficult than transforming laboratory strains of E. coli K12; the methods developed for K12 will not necessarily work for other strains or species. Electroporation is a more generally applicable method, though by no means a panacea. Note also that your cells are likely to contain restriction enzymes. The upshot of this is that while E. coli DH5 derivatives may give you millions of transformants per µg, natural strains are more likely to give you ten or none. You will need plasmid DNA in amounts of tens to hundreds of micrograms to have any chance of success. Good luck, and if you don t succeed, remember that you are in good company. Researchers struggled for decades to get E. coli to transform. View0 Recommendations

Abdurrahman Aygülasked a question related to PlasmidsIsn t plasmid stability an issue for therapeutic protein synthesis on an industrial scale?Question2 answersJul 10, 2021If we consider, for example, heterologous insulin biosynthesis in E. coli, we often find that a strategy based on synthesizing the target protein by expression plasmids has simply been adopted. But wouldn t that be a problem in terms of plasmid stability during continuous production on an industrial scale? Shouldn t such operations be performed with modifications at the chromosome level? There are definitely different nuances in other hormones, enzymes and proteins, thus I would appreciate it if you could explain by giving some examples.Thanks in advance..Relevant answerAbdurrahman AygülJul 12, 2021AnswerDear Pierre, thank you very much..View0 Recommendations

Yasu Xuasked a question related to PlasmidsPromoter of rop from plasmid pBR322?Question3 answersApr 24, 2021I am trying to locate the promoter of Rop gene on this plasmid. Is Rop co-expressed with TetR?FYI- It is from NEB.I want to extract the ori and rop to put in another plasmid to get a copy number ~20.I also looked at addgene . They mentioned a pColE1 with ~20-25 copies but I actually didn t find this plasmid. not sure about whether it is this one: ve looked they are both the ColE1 family, does it mean they both regulated with similar mechanism with Rop?Relevant answerSergey RykovJul 12, 2021AnswerI ve the same problem. I want to design shuttle vector with copy number in E.coli about 20 per cell. Alignment of pBR322 and pET-series plasmid give a ~1800 bp region, but i see it may be reduced to 1200-1300 bp fragment. I cant find any special works, where ColE-replicon from pBR322 get reduced in size with no changes in copy number.View0 Recommendations

Paikwin Liasked a question related to PlasmidsCould missense mutation lead to altered expression of the gene per se? What may be the mechanisms behind this? Question11 answersJul 9, 2021I m working on a certain protein (protein A). It has been constructed on a plasmid, and two point mutations have been created (WT and mutant 1 and 2). After transfecting cells with equal amounts of plasmid DNA, I found that gene expression of protein A in WT is twice that of mutant 2 and slightly higher than that of mutant 1. I ve repeated the same experiment 4 times and got the same result. I tried another batch of plasmid newly extracted fro E. Coli., same result. Is this a common phenomenon? What would be causing this? Is it meaningful to find out why and if so, how can I step by step figure it out? Relevant answerPaikwin LiJul 11, 2021AnswerNarumi Uno , thank you for your input! Best regards!View3 Recommendations

Sourav Dasasked a question related to PlasmidsLac operon mechanism hinderance during multicopy plasmids?Question1 answerJul 10, 2021However, when present in multicopy plasmids, lac promoters suffers from the disadvantage of sometimes having unacceptably high levels of expression in the absence of inducer (a.k.a. “leakiness”) due to titration of the low levels of the lac promoter repressor protein LacI from the single chromosomal copy of its gene (about 10 molecules per cell. Please Explain, what does it mean?Relevant answerHanna AlalamJul 11, 2021Answeryou normally have 1 copy of the lac promoter and 1 copy of the lac repressor (lacI) on the chromosome. The amount of repressor in enough to control the expression from the 1 copy you have on the chromosome, however, when you have a the lac promoter on a plasmid then suddenly there is much more promoter than the repressor can bind to. Think about it this way, assume you have 10 molecules of lacI which are sufficient to suppress 1 lac promoter. Now you have 20 lac promoters hence you require 200 molecules of lac to control it but the chromosomal copy only produces 10 and these will bind to the excess of promoter hence titrated when the lacI molecules bind 1 or 2 promoters the rest of the promoters that do not have lacI bound to them become active in absence of the inducer (IPTG or lactose). To overcome this many plasmid encode a copy of lacI, in this case when the lac promoter copy increase so does the copy number of the repressor (see pet16b for an example plasmid). Alternatively a single copy of chromosomal lacI can be sufficient to control expression from a multi copy vector if the promoter of lacI is mutated to produce higher than normal amounts of the protein (see lacIq1 promoter, with ref added below).ref: Recommendations

Mark K Cheeasked a question related to PlasmidsWhat plasmids can I use for fluorescent labeling of lactic acid bacteria?Question4 answersJun 23, 2021I am interested in labeling Lactobacillus plantarum with either a red (preferably) or a green fluorescent protein. Can anyone recommend a paper that describes a molecular tool for doing this? Thank you for your help.Relevant answerFrank R BurnsJul 9, 2021AnswerHi Mark,The authors of this paper may be able to provide you with one of their constructs, if not how to make them and the vectors to start with are well described.Article Use of green fluorescent protein to monitor Lactobacillus pl... FrankView5 Recommendations

Michael Plankasked a question related to PlasmidsDoes anyone have a recommendation for yeast reporter gene plasmid/protocol?Question3 answersMar 2, 2021Hi,could anyone recommend a plasmid and/or protocol for reporter gene assay in S. cerevisiae? I want to assess the effect of growth conditions on a transcription factor, so I want to clone it`s recognition sequence in front of a minimal promoter followed by reporter gene. Preferentially, I would use a luciferin or lacZ reporter.Thank you.Relevant answerMª Angeles Zorrilla Lopez-PereaJul 8, 2021Answer(it sounds similar to Y2H).?....View0 Recommendations

Jose Enrique Gonzalez-Pradaasked a question related to PlasmidsHas anybody had purity issues when cloning DNA in the pBI-CMV1 vector?Question3 answersJul 5, 2021Hi, I work with voltage-gated sodium channels, which are a pain to clone into high-copy number plasmids. Recently I have acquired the pBI-CMV1 vector, which is a low-copy number vector, and it has really helped with the cloning. However, when it comes to DNA purification, I have had lots of issues. Sometimes the DNA yields are extremely poor (I mean barely detectable by Nanodrop), despite longer growth periods and increased culture volumes/lysis buffers (Qiagen) compared to high-copy number plasmids. What is most worrying is that I often get a high level of smeariness when I run miniprep/midiprep pBI-CMV1 DNA on an agarose gel. It s not one distinct form of contamination; it s more of a background smear/smudge that stretches across all DNA sizes on the gel (and this can be seen even without adding restriction enzymes). It makes Nanodrop yields deceptively high and ruins the DNA for downstream applications like transfection. I m not quite sure what s causing it, as I ve never seen it with my other cloning/expression plasmids. I do not use endA+ bacterial strains for cloning. Has anyone worked with pBI-CMV1 before? Or has anybody encountered a similar problem with a different plasmid and been able to resolve/minimise it?Thanks!Relevant answerHanna AlalamJul 6, 2021AnswerHi Jose Enrique Gonzalez-Prada it just a matter of definition as what is their cutoff for low or medium. Upon further inspection of this plasmid I noticed there are two cmv promoters in it. Some viral promoters (such as CMV) do have an activity in E.coli check ref below. Plasmids that have active promoters without a proper bacterial terminator in it tend to produce lower yield (at least in my hands). As I said before extensive growing or up scaling the reaction might lead to increase genomic DNA contamination if your extraction. As an alternative approach try to do multiple mini-preps pool then ethanol precipitate (redo the transformation just to be sure). Hope this helps.ref:Article Viral promoters can initiate expression of toxin genes intro... View0 Recommendations

VENKATA SRI KRISHNA KONAasked a question related to PlasmidsWhat will be growth rate of BL21(DE3) with and without PET Vector plasmid in M9 media? Question1 answerJul 4, 2021 Without Induction i would like to know what will be growth rate in PET vector if transformed in BL21(DE3) ...By addition of 30mg/lit of Kanamycin in M9 media what will be relative effect ...Any data or research publication would help me in great way Relevant answerChristopher KesthelyJul 5, 2021AnswerFrom previous work, I would suggest that you shouldn t be able to notice any difference between the empty cultures and those containing the uninduced plasmid (should also be very close when induced provided that you are not expressing a toxic protein). As for the growth rate, Timing the start of division in E. coli: a single-cell study published in 2008, Reshes et al. suggest roughly 38 minutes for replication in M9.View0 Recommendations

Mara Llamasasked a question related to PlasmidsCan I transfect already stable transfected cells?Question5 answersJul 2, 2021Hi! I have a stable TCR transfected cell line but I realized I should also express CD8 alpha and beta chains in order to properly test antigen response. One option is to go back to plasmid production and ligate my TCR chains into plasmids already containing CD8alpha or CD8beta chains, and then transfect cells with them.But since I already have the TCR expressing cell lines, I wonder if I can just transfect CD8 alpha and beta chains into them. Has anyone had any similar experience?Relevant answerMara LlamasJul 5, 2021AnswerThank you very much for your answers. Yes, Narumi Uno I need to express CD8 for complexing with HLA and proper signaling to T cells. But I just recognized one more problem and it is that my original vectors already supply a truncated form of CD8, and we actually use this for sorting transfected cells, but it is not functional. So even if I add the functional one in a second transfection, I will not be able to distinguish cells with truncated CD8 from the ones with full CD8 :(But thank you everyone for the discussion! View3 Recommendations

Yue Qinasked a question related to PlasmidsHow to get overexpressed HLA-I with correct subcellular localization?Question1 answerJul 2, 2021I constructed overexpression plasmids of Class I MHC molecules (HLA-I), with C-terminal Myc-FLAG tag or GFP-tag. I transfect the plasmids in 293T cells to test expression. The expression can be detected by Western Blot. But the subcellular localization is mainly in cytosol. Flow cytometry result showed less than 3% positive of surface staining of the HLA antibodies, respectively. Then I co-transfected the plasmids with B2M plasmid, but still low surface expression. The HLA-E flow antibody can detect endogenous protein in other cell lines. I think the problem should be the plasmid.Any one has suggestion or experience with this issue? Thanks!Relevant answerNarumi UnoJul 3, 2021AnswerDear Yue Qin,I m not an expert in HLA.I wonder if the tag at the C-terminus inhibits the antigen presentation of HLA-E, and thus prevents it from migrating to the cell surface.Why not try expressing untagged HLA-E?Best,NarumiView0 Recommendations

Nicole Belangerasked a question related to PlasmidsTrouble digesting supercoiled plasmid into linear form?Question5 answersJun 29, 2021I used the TOPO TA Cloning Kit for Sequencing to clone a desired gene into the pCR®4-TOPO® vector. I have been able to confirm the presence of my gene by colony PCR. I then extracted my plasmids using Qiagen Midi Prep Kit. I did quantify them so the extractions are pure. When I run them in an agarose gel (1%), you can see my isolated plasmid is super-coiled. I want it in linear form so I am just cutting at 1 site and I have tried using the following enzymes: NcoI, EagI, and EcoRI (none of which will cut my insert). All of these enzymes are failing to digest, whether I do it for 60 minutes, 90 minutes, or overnight. I should only have 1 band around 4.1kb but am getting these results instead. Can anyone help to solve the issue?Addditional Information:Digestion is done in thermocycler to ensure consistent temperatureUsing 1uL NcoI and 1ug of DNAI have tried several extraction kits: Qiagen Mini, Midi, Maxi and ZymoMiniNcoI Digestion.pdf416.18 KBRelevant answerRui ChenJul 2, 2021AnswerFirst, you must confirm that your fragment has been inserted into the vector backbone. This problem is well solved, that is, the extracted positive plasmid and the empty vector are electrophoresed together, and the gel concentration is 0.5%. During this period, it should be observed that the empty vector migrates quickly. Or perform sanger sequencing. Secondly, there is only one band of the fully cut vector, and depending on your results, it does not rule out incomplete digestion. Third, the enzyme has a fixed-matched enzyme-cut buffer, I don t know if you have considered it.View0 Recommendations

Andrés Castilloasked a question related to PlasmidsStrange sgRNA scaffold in Addgene plasmids?Question1 answerJun 29, 2021Sorry for my ignorance, but I am having difficulties to understand why in the plasmid pHSN6A01 (, between the pol III promoter (AtU6-26) and the gRNA scaffold, there is a sequence which carries antibiotic resistance (SmR). I can not find the sense of it as the Pol III could not transcribe the resistance gene and all of the space would be negative for the sgRNA generation.Relevant answerFrancesco AulicinoJun 30, 2021AnswerHi Andres,The SmR is used as a stuffer. You need to digest the plasmid with BsaI (double cutter) to insert your sgRNA. The SmR is only useful for propagation (ensure that the cloning cassette is not lost) and counter-selection (+ clones should not grow on Spec). Details for the cloning method are found in the original publication, this is a common cloning strategy for sgRNAs using annealed oligos cloning.Hope it helpsFrancescoView0 Recommendations

Harpreet Sangehasked a question related to PlasmidsPlasmid for CRISPR in plants?Question1 answerJun 24, 2021Hi all. I am looking for a plasmid to do a gene knockout in Arabidopsis thaliana. I would like to have a fluorescence gene (RFP/GFP), antibiotic resistance (kan/amp/sm), Cas9, At promoter and also cloning site for sgRNA. Could anyone please guide me in choosing the plasmid as I have been stuck at this point for more than a month?Relevant answerAmy KlockoJun 29, 2021AnswerHmm, that is quite the plasmid, I have not seen one with all those features. Why the GFP/RFP? It s more typical to find a vector with a plant selection marker (hyg/kan/bar) rather than a screenable marker.You may need to obtain a more standard vector then modify it for the features you want.Check for publications using CRISPR in plants, see if one of those vectors looks suitable, then ask the authors if they can share their vector (or order from the same place they did if it s a commercial one)View0 Recommendations

Mercedes Vazquezasked a question related to PlasmidsAny suggestions on my problem with electrotransformation of pE-FLP (flippase expression vector)?Question6 answersMay 21, 2015Hi, I m trying to disrupt some genes of the genome of E. coli. For achieving this, I m following the protocol of the P1 phage in which this phage transfers genetic material from one strain to another. The recipient strain is from the keio collection, so I don t have any problem with the selection of the colonies which were succesfully trasduced.But after the confirmation of the disruption with the antibiotic screening and PCR, I tried to remove the Kn resistance using flippase. The flippase is a recombinase that recognize FRT (Flippase recognition target) sites and removes all the flanked area.This plasmid must be electroporated and then grow it at 30°C, because it has a temperature sensitive ori.I have done all of this but I have not obtained any transformant. Do you have any clue of why I don t get colonies? Can you give me some tips?Relevant answerOsama KamalJun 29, 2021AnswerMercedes Vazquez Can I as you what plasmid you are talking about? Is it pCP20?Many thanks?View0 Recommendations

Gabriela Guevaraasked a question related to PlasmidsSUMO tag needs a SUMOpro Fusion Tag? Question3 answersJun 28, 2021I would like to used to use sumo protease. However, I did not find the sequence that the protease recognizes in order to order a plasmid with this sequence? and the information available on the internet using these sequences for many other purposes that make me confused about the function of this protease as a protein fusion domain ?? Anybody has ever used the SUMO as a cleavage protease? it is someone who did could you please share with me the recognition sequence or the paper, please. Thanks Relevant answerHüseyin BesirJun 28, 2021AnswerThe SUMO-tag/SUMO protease system is special because it does not have a particular short recognition sequence (like other proteases) for the protease but the whole SUMO protein is needed to make the protease cut at the Gly-Gly-site at the end of SUMO right before the first amino acid of the target protein (can be any amino acid except for proline). You can contact the EMBL Protein Expression and Purification Core Facility where you can get an expression vector.View5 Recommendations

Muhammad Haider Farooq Khanasked a question related to PlasmidsIdeal Plasmid or vector for in vitro mRNA synthesis?Question3 answersJun 10, 2021I want to clone my gene of interest in a vector/ plasmid that can be used for in vitro mRNA synthesis. Which commercially available vector is usually used for in vitro mRNA synthesis using T7 promoter-driven transcription? Is there any vector available with polyA tail, 3 and 5 UTR of alpha or beta globin with a cloning cassette that can be used to insert the CDS? I can also utilize any other UTRs which are reported to enhance mRNA efficiency/ stability.Relevant answerShruti ChitranshJun 28, 2021AnswerWe added them through primers in the CDS and then cloned in the vector. View0 Recommendations

Dat Nguyenasked a question related to PlasmidsWhere to find the map for pHH21 plasmid?Question1 answerJun 24, 2021Hi all,I wonder if anyone know where I can find the plasmid map for pHH21 that is commonly used in Influenza research?Relevant answerMalcolm NobreJun 27, 2021AnswerHello Dat Nguyen Please refer to the link below. hope this helps.Best.View0 Recommendations

Ana Patricia Ramosasked a question related to PlasmidsSURE competent cells and CCDB resistance - can anyone help?Question7 answersAug 5, 2014Hi all.For my last LR reaction I used SURE competent cells cells (link) for the transformation (since the final plasmid was very big).I got many colonies (which is always suspicious) and end up finding that most (if not all) had the pdest vector (wich has the CCDB lethal gene). I finally transform 3 different destination vectors in SURE cells and DH5alpha and got to the conclusion that SURE cells seems to be resistant to the ccdb gene.This resistance is not reported by the company. Does anyone has experienced with this type of competent cells? Is it normal this resistance?Thanks tabId=AG-PR-1218Relevant answerSyed Adeel ZafarJun 24, 2021AnswerHello Guys, can we use DH5a cells for gateway reactions (BP and LR) which release the ccdB gene? I am afraid as I cant see any info about CCDB resistance of DH5a cells, and user guide says we must use ccdB resistant cells. Please answer. Thanks Ana Patricia Ramos Michael J. Benedik Tomas Santalucía Xiao Zhou Alejandro Martin View0 Recommendations

Vanessa Sewellasked a question related to PlasmidsMaxi plasmid prep issues - using a different vector?Question6 answersJun 24, 2021Hello.I have been doing Maxipreps with plasmids in a pUC19 vector and they worked great! However, when I try to do a plasmid prep for another plasmid, they are failing. I am not getting ANY DNA.I am using the same protocol - streak on plate, pick a colony, grow in 5 ml LB, then inoculate 250 ml of 2YT with 2.5ml of the starter culture. I grew this overnight at 37 degrees and 200 rpm.My GOI is in a pET30 backbone and includes a 10xHis, 3xFLAG and TEV site.I should also note, that when I did a mini prep, it worked (however, it was not supercoiled plasmid, this might be due to old columns)ANY recommendations would be greatly appreciated. Thank you.Relevant answerHanna AlalamJun 24, 2021Answer I think your getting over saturated cultures especially since you are using 2xYT. This will lead to clogging of columns. Dilute 1:1000 and not 1:100 and grow at 30C. It could be that your cells grew more slowly with pUC19 since it has a much higher copy number than pet30 and hence were not as saturated. For exact O.D harvest the cells as indicated in your kit s manual and if you keep getting problems you might want to switch to LB instead. View2 Recommendations

Alka Senwalasked a question related to PlasmidspEC86 plasmid map?Question3 answersJun 22, 2021Hi everyone, Does anyone know which inducer is needed to overexpress pEC86 plasmid ? Has anyone used this plasmid to overexpress cytochromes and has details? ThanksRelevant answerAlexandra JohnsonJun 23, 2021AnswerI uploaded the original paper it s from, if I m reading it correctly pEC86 isn t an overexpression plasmid, it s a helper plasmid and a separate overexpression plasmid is needed for actually expressing the cytochrome of interest. pEC86 constitutively expresses the cytochrome maturation genes and doesn t need to be induced. Here s a map: which also says the same thing.Biochemical and Biophysical Research Communications Volume 251 issue 3 1998 [doi 10.1006_bbrc.1998.9549] Engin Arslan; Henk Schulz; Rachel Zufferey; Peter Künzler; Lind -- Overproduction of theBrady.pdf220.98 KBView2 Recommendations

Phuong Nguyenasked a question related to PlasmidsHello, I want to ask a question about DNA vaccine?Question3 answersJun 23, 2021I m going to make DNA vaccine for fish by using plasmid pcDNA3.1(+) contains a bacterial antigen, as you may know for this plasmid to make an expression protein we need to add a Kozak sequence as a RBS to star the translation,unfortunately, in my target gene contains another Kozak sequence at the middle of the gene, which maybe will cause unwanted, my question is should I still use this gene of interest (bacterial gene) ?Thank you for your reading, Relevant answerPrescott DeiningerJun 23, 2021AnswerYou can certainly use it because translation will primarily default to the first decent Kozak sequence. If you are concerned, you can mutate the internal Kozak using a silent mutation. It wouldn t be bad to just resynthesize your bacterial gene with the optimal codon usage and eliminate things like the internal Kozak while you are doing it. It doesn t cost that much to have things synthesized.View12 Recommendations

Mkhuleko Singwaneasked a question related to PlasmidsWhat controls to include for transient transfection of plasmid DNA in a mammalian system?Question6 answersJun 20, 2021Hi,I am trying to transfect plasmid DNA in HEK293 cells, I m trying to figure out what positive control I can use to be sure that the cells are able to express my protein and if my transfection method works or if there was successful transfection.I would also appreciate some tips on a negative control too, but I figured I will not not include the plasmid/DNA in my negative control just the Transfection Reagent, cells and serum free media.Thank you...Relevant answerTombari Pius MonsiJun 20, 2021AnswerI think you are on the right track. I did a transfection of MRP4 DNA plasmid into HEK 293 cells using PEI protocol. In transfection, a researcher is trying to express a particular gene in a cell. This means the cell line to be transfected do not have this gene or has it at a very low level. I used HEK 293 treated in only DMEM as my negative control. A positive control should ideally be a cell line that naturally express the gene to be transfected or commercially available cell line that has your gene of interest. In my opinion, a positive control is not very vital because you can always use a purchased purified protein of the gene of interest to run alongside your transfection products on SDS-PAGE prior to western blotting. This tells you if your transfection worked.You can also use a real-time PCR to quantify the level of expression of your gene. It will be ideal to optimise your transfection protocolView3 Recommendations

Rahul Jainasked a question related to PlasmidsAny tricks to increase conjugation frequency in Gammaproteobacteria using Ecoli S17-1 donor harboring Tn5 based fluorescent plasmids?Question1 answerApr 26, 2021I am trying to label some of the bacterial endophytes (Pseudomonas, Serratia, Pantoea, Acinetobacter, Sphingomonas and Arthrobacter) for performing in-planta studies.I have chromatic plasmids including (pMRE-Tn5-132; pMRE-Tn5-133; pMRE-Tn5-135; and  pMRE-Tn5-155) but I am not getting transconjugants with the successful integration of fluorescent genes except for Pseudomonas-mRE-Tn5-155. Any help or tricks to increase the conjugation efficiency!!Relevant answerRiaz A. KhanJun 20, 2021AnswerChemically assisted surface conjugation, linker, or incubation at optimum conditions can be tried.View0 Recommendations

Amanda Lovecraftasked a question related to PlasmidsSingle copy of TetO (Tet operator) for bacterial expression?Question1 answerApr 21, 2016I have a plasmid map and a plasmid which I ve sequenced, and I found a discrepancy where the map contains two copies of TetO separated by ~30bp whereas my sequencing result shows only one copy of TetO. I ve looked online but am having trouble finding any information about expression systems that use only a single copy of TetO. Most sources describe 2 or 7 copies. Can anyone tell me if this is viable for TetR binding? Relevant answerClément BaretJun 20, 2021AnswerLate answer but might be useful for someone else:One TetO box corresponds to a pXyl/Yet promoter. It can sufficiently block expression with very low basal expression. Make sure your -35/-10 boxes are still present in your design.Source :10.1111/j.1751-7915.2007.00001.xView0 Recommendations

Marine Amouriqasked a question related to PlasmidsWhat is the strange band for my native plasmid on electrophoresis agarose gel?Question4 answersJun 18, 2021Hello,I made an electrophoresis gel with the same plasmid (5828kb) but with:- 2 different concentrations:-in well 2-5; 0.5µg of DNA-in well 3-4-6-7; 1µg of DNA- 2 different manufactured batch of plasmids (perform by 2 different suppliers):-in well 2-3-4: first suppliers-in well 5-6-7 the second suppliersI have the nicked form (second band) and the supercoiled form (third band). But I don’t know what is the first band. This band more important with the first supplier batch. Can you help me to explain this band?do have any explanation?Moreover, my A260/A230 ratio is a little too high (2.3) but I have the same result in both suppliers. do have any explanation to explain it? What could be the impact on my electroporation efficiency?Thank for your help

electrophoresis gel.png141.81 KBRelevant answerAndrew J SpiersJun 18, 2021AnswerDear Marine,Identifying plasmid bands from uncut DNA samples on a gel is always a problem, and depending on the isolation protocol, age and storage of the sample, you can have more nicked (relaxed) and supercoiled forms, along with degradation smears. I find it better to check the quality of my plasmid preparations by linearising them with an appropriate restriction enzyme - or even cutting them in to a series of fragments - and then looking at the results on a gel of the appropriate gel concentration.If you know the restriction map of the plasmid, you have a clear idea of the bands you expect to see - and if they are nicely within your DNA ladder, you can determine sizes easily. Contaminating DNA will be obvious too (degrade DNA may disappear during the restriction incubation though). AndrewView4 Recommendations

Ahmed Abdelrahmanasked a question related to PlasmidsWhat is the best method (transfection reagent) for transfection of Human liver sinusoidal endothelial cells and HUVECs as well-based on experience?Question2 answersJun 17, 2021I try to transfect endothelial cells with firelfy luciferase plasmids under control of various promoters with Lipofectamine LTX, but transfection does not work at all, does any one have any idea how to transfect these endothelial cell lines ? Relevant answerAhmed AbdelrahmanJun 18, 2021AnswerThanks VeraView0 Recommendations

Weibin Zhouasked a question related to PlasmidsProblem in making a lentiviral contruct?Question3 answersJun 15, 2021Hi, I am trying to making a lentiviral construct for expressing a gene of our interest in mammalian cells. I tried to use two RE sites to replace the CDS that is already in the lenviral construct with the one I want to express.The problem I have is that after transforming Stbl3 cells with the ligation product, I got a decent number of colonies, but none of them contain my gene of interest. Also, I am not able to use sequencing primers such as EF1a-F to sequence the plasmids that I extracted from those colonies. Is there any special procedure to handle the lentiviral construct? Do I have to grow the cells at 30 C? looks like Stbl3 can be grown at 37 C though. thanks .Relevant answerShruti ChitranshJun 18, 2021AnswerLentiviral constructs has LTR elements and that s why they are unstable and tend to recombine given favorable growth conditions. Use lower temperature and very low rpm with increased regeneration time post transformation in STABL cells. This will lead to more positive clones with less recombinations. Also I agree upon picking small/medium size colonies and 1:3 ratio for ligation. View0 Recommendations

Sam Clemensasked a question related to PlasmidsIs it possible to isolate 200kb plasmid with standard alkaline lysis technique?Question3 answersJun 15, 2021The bacteria contains two megaplasmids, one of which is 233kb. Using a Qiagen miniprep kit with spin columns, is it possible to elute even a small amount of DNA this large? Are there any modifications to be made which do not involve obtaining the anion-exchange kits designed for large constructs?Relevant answerShruti ChitranshJun 18, 2021Answerlyse and neutralize your plasmids using kits reagents then take the supernatant and perform phenol chloroform extraction followed by ethanol precipitation and get pure DNA extracted of any size.View6 Recommendations

Wesley Hansonasked a question related to PlasmidsExperiences using Gibson assembly for cloning into large constructs?Question3 answersJun 16, 2021I am attempting to clone a mutant gene into a large (~22 kb) plasmid containing a viral genome and just recently realized that the established method of restriction cloning that was used successfully in the past will not work in this instance due to the location of one of the cut sites. My immediate thought is to use Gibson assembly and forego restriction enzymes altogether, though I am hesitant because I don t have any experience using Gibson in such large constructs. Does anyone have any experience with this or similar situations (large expression vectors, BACs, etc.)? Any contributions are helpfulRelevant answerAndrea GuedezJun 17, 2021AnswerI used Gibson assembly once for my plasmid but I was trying to generate a library of compounds and I did not get enough efficiency, but I did get some clones. You can also try some overlapping PCR steps to generate some of the inserts and then, RD/ligation. View0 Recommendations

Janine M LeBlanc-Straceskiasked a question related to PlasmidsTransforming a resistant bacterial strain with plasmid with same resistance gene?Question2 answersJun 15, 2021I have a chloramphenicol resistant expression vector that I want to transform into Rosetta (DE3). Both the expression vector and the host cell (that carries a plasmid with tRNA genes) are chloramphenicol resistant. If I increase the concentration of chloramphenicol can I select for cells that have taken up the expression vector? Relevant answerMichael J. BenedikJun 17, 2021AnswerI have to disagree, I don t think you will be able to make that work. Plasmids are already quite high copy number so going 2x higher is not going to change your level of drug resistance in any practical measure. In fact even one copy on the chromosome confers resistance nearly indistinguishable from a plasmid. You really need to have one of your plasmids with a different selective marker. View10 Recommendations

Fabiola Pulidoasked a question related to PlasmidsHow can I account for gene copy number in qpcr biomass measurements (fungal community)?Question1 answerJun 13, 2021Hi everyone, I performed qPCR analysis using the FungiQuant primers to target the 18S region for fungi to determine fungal biomass. I used a plasmid standard containing this region from S. cerevisiae and was able to get some biomass measurements. However, I am unsure how to account for variation in gene copy numbers. If anyone has any ideas or can guide me to an article, I would greatly appreciate it. Relevant answerAbhijeet SinghJun 16, 2021AnswerSimple answer: you can t.Complex answer: it requires details of all fungal species present in your sample and information about each genome and gene copy number, followed by some calculations to correct for copy numbers. Unless you have all these things, you can t correct for copy number variation in your analysis. View0 Recommendations

Aadil Javedasked a question related to PlasmidsWhile inserting GOI in pCW57.1 lentiviral plasmid DNA, is it important to delete ccdB gene fragment?Question3 answersJul 18, 2020I am facing a problem with the plasmid from addgene (pcW57.1), when transformed into chemically competent DHFa cells, did not give any single colony overnight in ampicilin containing agar media. am planning to remove ccdB gene by Nhe1 and Sal1 cuts and then by blunt ended ligations I will transform again. Will this be correct approach? (Later I will insert our GOI with blunt ends too)..Kindly anyone with experience of working with this plasmid and or system, please share your opinion.Relevant answerAadil JavedJun 14, 2021AnswerI am reading this quite late. I was blunting after digests with T4 polymerase. The cloning worked afterall, I have the inducible expression system in our lab :) Thanks.View0 Recommendations

Oliyad Jeiluasked a question related to PlasmidsIs the Plasmid DNA Miniprep Kit work well for extracting fosmid DNAs?Question4 answersJun 7, 2021I was extracting fosmid DNA from the ecoli clone and extracted the fosmid using is the Plasmid DNA Miniprep Kit. However, the extract fosmids are less in quantity and not qualified for sequencing. Thus, is the Plasmid DNA Miniprep Kit work well for extracting fosmid DNAs? or any other alternative? Relevant answerAlexandra JohnsonJun 11, 2021AnswerThe F plasmid origin in a Fosmid is going to replicate at a much smaller copy number than most plasmids you ve probably worked with, there s only going to be 1-2 per E. coli cell. For comparison the low copy p15a origin that s pretty common in labs has a copy number of around 10-20. It s normal to get very low yield from a miniprep of these plasmids, even without an insert. If you have a large insert the yields will be even lower. Some people do maxipreps of these and concentrate them after elution to make up for it, especially if they need them at concentrations for sequencing. Some BACs/Fosmids contain a 2nd, higher copy origin which is inducible to avoid that. View6 Recommendations

Gustav N Sundellasked a question related to PlasmidsCan you search for standard primers in a DNA sequence? or standard primers as multiple fasta?Question3 answersJun 10, 2021Hi Is there an online tool to search for standard primers in a DNA sequence? I did a fast google and i didn t find any. i want to upload a plasmid or any DNA sequence and it automatically maps standard primers on to it. Alternatively do anyone of you have a file with standard primers in a multiple fasta or other multiple sequence file? Relevant answerMichael J. BenedikJun 10, 2021AnswerYou could just make a text file with all the standard primer sequences you have in your lab in Fasta format and just search against that with each plasmid you might have. It probably will take less than an hour of time to create and then you are good to go and you can always add to it anytime. View0 Recommendations

Stepanka Nedvedovaasked a question related to PlasmidsDoes anyone know the pLEXSY-sat2 (EGE-234) sequence ?Question1 answerJun 9, 2021I have an older version of the current plasmids at my disposal. Unfortunately, it differs by 562 bp from today s pLEXSY-sat2.1. And unfortunately, I can t find the original sequence anywhere. Does anyone happen to have this sequence? Thank you! Relevant answerHenry KolgeJun 9, 2021Answer log$=nuclalign blast_rank=1 RID=C1PE3NVC013is this the one?Plasmid seq.txt9.62 KBView0 Recommendations

Akshay Tawaleasked a question related to PlasmidsWhere I can get good plasmid backbones for B subtilis for expression purpose?Question6 answersJun 7, 2021Here I am trying to express the metalloprotease in b subtilis using my interested promoters, which is not homologous to that organism, i.e., a human metalloprotease. But I didn t get any satisfactory plasmids to do so.So anyone suggests where I can get such plasmids?Relevant answerAkshay TawaleJun 9, 2021AnswerOkay, thank you, Manuele Martinelli I ll check.View0 Recommendations

Aulia Fitriasked a question related to PlasmidsThe protein can not expressed in Western BlotQuestion3 answersJun 7, 2021Dear all researchers, I have a question. I made some plasmids with HA-tag, and one of them has successfully expressed in Western Blot. However, the others plasmids couldn t be expressed by Western Blot, even though I have been checked their sequencing analysis and there was neither frameshift nor mutation. I made protein preparation with RIPA lysis buffer and make denaturation with 95C in 5 minutes. I also make different gel SDS-page according to their molecular weight. But when I try detection with HA antibody, the protein of my plasmids couldn t be expressed. Is there any suggestion? Thank you very much for your attention.Relevant answerPapri BasakJun 8, 2021AnswerMaybe you should try using different induction conditions and check their effect on the protein expression. I would suggest first check the intensity of the expressed protein by coomassie stain. Loading crude lysates may be confusing, it would be better if you load purified samplesView0 Recommendations

Jingfei Zhuasked a question related to PlasmidsHow to cotransfect plasmid and RNA molecule?Question3 answersMay 18, 2021Hi all,I would like to cotransfect plasmid DNA and 700 nt RNA molecules in 293T cells, and tried lipo2000. But I don t know if it is an efficient regent for my project. Is there anyone who has this experience and give me some advice?Thank you very much~Relevant answerJunjie ShaoJun 8, 2021Answerif your RNA is purified mRNAs, that should be ok. try it first.some studies have shown T7 transcribed RNA sometimes contains RIG-I ligand.View0 Recommendations

Reina Kiyonoasked a question related to PlasmidsWhy transformation efficiency decreases? Question3 answersJun 4, 2021It is well known that transformation efficiency decreases by addition of excess amounts of plasmid. I would like to know the molecular mechanism. What factors do you think affect it?Thank you for your help. Relevant answerJohn SchloendornJun 6, 2021AnswerYou end up putting multiple plasmid molecules into each cell. The mechanism is purely statistical distribution. You can even calculate it, if you count the number of cells available by plating without selection, and do the math to figure out how many plasmid molecules you ve got. You can affect it by packing more cells (at greater density) into your competent cells preparation. That will of course work only for so long. I think it s one of the reasons that electrocompetent cells are so much more efficient than chemically competent cells -- you can pack them more densely, by a considerable factor. View4 Recommendations

Beatriz Gonzalezasked a question related to PlasmidsHow to make stable EGFP and mCherry stable cell linesQuestion1 answerJun 1, 2021Does anyone have a recommendation for a good plasmid to stable transfect mammalian cell lines with fluorescent proteins such as EGFP or mCherry? ThanksRelevant answerNarumi UnoJun 4, 2021AnswerHi BeatrizWe recommend that you use constitutive expression vectors like pCAG-GFP (Plasmid #11150) and CAG-mCherry (Plasmid #108685).With CAG promoters, the genes are highly expressed in a wide range of mammalian cells.Stem cells, such as pluripotent stem cells, are considered to be lost in expression, so an EF1alpha promoter such as pEF-GFP (Plasmid #11154) is a good EF1alpha promoter. A CMV promoter may also be sufficient for stable gene expression.I usually insert drug resistance genes (Neomycin resistant gene) to these vectors to produce stable expression strains by drug selection.BestView0 Recommendations

Yeon Soo Jeonasked a question related to PlasmidsHow to get the plasmid from AddgeneQuestion4 answersJun 1, 2021I would like to purchase a general GFP reporter plasmid from Addgene but it is not available for the profit organization. Could anyone please give me advice how to deal with this problem?Relevant answerAmy KlockoJun 3, 2021Answercontact their customer service, if there are restrictions on commercial use of their vectors they could explain more about what your options areView4 Recommendations

Felipe Del Valleasked a question related to PlasmidsWhat s a good plasmid that marks plasma Membrane for B Cells?Question2 answersNov 9, 2019Hi everyone ! I m looking for an expression plasmid (hopefully with a fluorescent tag) for observing plasma membrane changes that work properly in B cells, this since I m planning to acquire videos of this phenomena. I ve found a lot of references for TULP family plasmids (TUBBY-EGFP for example) that works well in other cell models. Can you suggest any? Thank you in advance.Relevant answerInna GoliandJun 2, 2021AnswerCAAX-GFP is a great plasmid for this purposeView0 Recommendations

John Albert M. Caraanasked a question related to PlasmidsIs EHA105 strain of A. tumefaciens resistant to spectinomycin?Question5 answersMay 24, 2021I ve been trying to transform my plasmid conferring spectinomycin resistance into EHA105 (via freeze-thaw method). Unfortunately, I ve been observing bacterial growth on the entire plate and not getting single colonies. I have tried to increase the concentration of spectinomycin, reduced the recovery time, and diluted the recovered cells before plating - same results. I am now planning to reduce the initial volume of the competent cells that I will transform (from 150 uL to around 35 uL).Relevant answerAlexandra JohnsonJun 1, 2021AnswerI did some literature digging and apparently part of EHA105 s construction involved transforming it with then curing it of a plasmid called pPH1JI. pPH1JI contains multiple drug resistances on what is probably a composite transposon, including resistance to spectinomycin gentamicin. Only loss of gentamicin resistance was verified, so my speculation is that some other resistance gene(s) could have transposed to either pEHA105 or into the chromosome unnoticed. That s assuming the strain you have is actually EHA105 though. A physical map of pPH1JI and pJB4JI.pdf243.55 KBNew Agrobacterium helper plasmids for gene transfer.pdf1.32 MBView10 Recommendations

Junhyung Cheonasked a question related to PlasmidsNo visible pellet after isopropanol precipitation using Qiagen midi kit?Question10 answersAug 22, 2019Hi I m trying to prepare plasmid dna for transfection but I m having difficulty getting dna from Qiagen midi prep. I not seeing a visible pellet after isopropanol precipitation and it is driving me nuts!! Other people using the same kit had good yields and I used the same Qiagen maxi kit and had a good yield so I think there is no problem with the kit. I m wondering if Dna yields are different with different plasmids? Am I just trying to prepare a difficult plasmid dna? I think I will try to proceed after the isopropanol step but I don t think it will have a good yield. Does anyone have the same problem? How did you solved it?Relevant answerMelike GezenJun 1, 2021AnswerMy problem is not about the yield more likely about a buffer or sth. The bacterial pellet is good like always. This is not the first time I am trying to isolate this plasmid thats how I know. Generally, I get around 2000 ug of DNA min. But I was curious this time ıf anyone else experienced the samething. This Qıagen Kit is the new box we just opened.View5 Recommendations

Volodymyr Segiovich Kavetskyyasked a question related to PlasmidsWhy doesnt my sequncing work with Forward primer ?Question3 answersMay 30, 2021I am having a problem with the sequencing of recombinant plasmids. I need to get sequences in both forward and reverse directions. For sequencing, I am using M13 Forward and Reverse primers separately. In 99% of cases, I get good sequences with the reverse primer. The sequences correspond to the expected size of my gene (approx 1000 b.p.). But when I try sequencing in forward direction I cannot get a full sequence. The fragments are approximately 150 b.p. I have attached the example of sequences that I received. Could anybody help with this?Thank you in advance Sequnces.rar194.10 KBRelevant answerPaul RutlandMay 31, 2021AnswerIf we make a contig of the 2 sequences M13r gives a weak intensity but very good clean and well spaced sequence from about 200-890 ( contig base numbers) getting much weaker in the bases below 200. This is normal sequencing and template and primer are correctly matched in concentrations. With M13F the sequence is very strong in intensity from 80-230 of the contig dropping off very quickly to give no analysable signal after base 230. This usually indicates too much template or too much sequencing primer in the sequencing reaction. Since M13R worked well we might assume that the amount of template is the same in both reactions and is correct so my guess is that you have too much M13F primer giving a very strong early signal but depleting the sequencing mix completely so you cannot get any long sequence. The solution is either use more sequencing reagent mix or less M13F primerView5 Recommendations

Mamta Vermaasked a question related to PlasmidsWhat vectors (Plasmids) are required for CRISPR/Cas9 mediated gene knock in?Question3 answersMay 11, 2021Hi, I am trying to tag a protein endogenously in Human iPSC model. While reading the protocols, I found different plasmids and I got confused which one works best for hiPCSs.Can anyone help me and share their experience?Relevant answerNarumi UnoMay 31, 2021AnswerI will answer late. I often do Knock out and HDR in human iPS cells. The following vectors are recommended for CRISPR / Cas9 expression. PX330-U6-Chimeric_BB-CBh-hSpCas9 provided by Zhang lab. You can purchase it from addgene. Since pX330 is not highly expressed in human iPS cells, Knock out is easy, but the frequency of homologous recombination is low. I have the impression that the Cas9 expression system by the EF1 promoter is superior. Please refer to this paper. On the other hand, in recent years, Cas9 and gRNA can be purchased directly as purified proteins and synthetic RNAs. I purchase from IDT (integrated DNA technology). Although expensive, they are simple and very efficient. It s a lot cheaper than making mistakes. Numerous detailed protocols are provided. The HDR vector has homologous recombination sequences (about 1 kb) corresponding to the cut site(s) on both ends, and drug resistance markers and fluorescent markers are placed between them. An EF1 promoter or CAG promoter should be used to express the marker protein. The CMV promoter will easily disappear.These are examples of introduction.gRNA expression pX330 vector: 2µgCircular fluorescent protein expressing-HDR vector: 8 µgHiPS cells (Feeder-free cultured): 3x10 ^ 6 cellsNEPA21 electroporater was used for gene-introduction.It is better to use the HDR vector as it is in circular preventing random integration of the HDR vector. Fluorescence expression will be observed from the next day. Transient fluorescence expression lasts for several days. It is possible to acquire DNA 48 hours after gene transfer and evaluate whether homologous recombination has occurred by PCR analysis. Fluorescence-positive cells are obtained by FACS one week later in order to obtain cells with homologous recombination. If you reply to this answer, you can have a more detailed discussion.BestView0 Recommendations

Jinlong Yuasked a question related to PlasmidsHow to select antibiotic resistance when constructing a plasmid ?Question7 answersMay 31, 2021Hi everyone, I am constructing a S. aureus/E. coli shuttle plasmid and was confused by how to choose antibiotic resistance genes. According to my observation, most of the S. aureus/E. coli shuttle plasmids contain two antibiotic resistance genes(ampicilin and chloramphenicol resistance). Here is my question:I want to know why many researchers use ampicilin to select for E. coli transformants and use chloramphenicol to select for S. aureus transformants. Can I just construct a plasmid with only one resistance gene (chloramphenicol) and use it for tranformant selection in both E. coli and S. aureus transformant?Relevant answerJaydip DattaMay 31, 2021Answer Recommendations

Anderson Javier Castillaasked a question related to PlasmidsDoes anybody have experience using a positive control for sequence amplification from P. aeruginosa colonies?Question2 answersMay 28, 2021Currently, I am struggling to verify the transformation of Pseudomonas aeruginosa with shuttle plasmids that I assembled in vitro and amplified in E. coli. Unfortunately, the colony-PCR experience in our lab is exclusively in E. coli, and while I try to adapt protocols which are recommended for P. aeruginosa, I can t get any amplification. At this point, and because other PCRs are running well, we think that we need a positive control for P. aeruginosa.  Maybe amplifying a gene-sequence located in the chromosome could be used as such?Does anybody have any experience using a positive control for amplification from P. aeruginosa colonies?Thanks in advance!Relevant answerAnderson Javier CastillaMay 28, 2021AnswerMany thanks for your recomendation !View0 Recommendations

Anastasiya Isaevaasked a question related to PlasmidsWhy am I getting low concentration of DNA plasmid after DNA extraction from clones?Question9 answersMay 27, 2021DNA is about 8kb, cells: XL1-blue, selection: ampicillin, PCR screening of clones: Encyclo Plus PCR Kit (Evrogen), DNA exstraction: Plasmid Miniprep (Evrogen).Site-directed mutagenesis per QuikChange Site-Directed Mutagenesis protocol using Thermo Scientific Phusion Hot Start II High-Fidelity DNA Polymerase kit.P.S. All kits work good (I ve got this DNA with another mutations).Relevant answerMimi AsogwaMay 28, 2021AnswerI agree with Khajezade Mohadese , reduce elution buffer, check antibiotic is not out of date and fresh Amp plates always used for selection. Check your kit is suitable for extraction of 8 kb plasmid, some kits are better than others. How low is the concentration?View10 Recommendations

Doina Turcanasked a question related to PlasmidsWorking with plasmid kits requires a special laboratory accreditation?Question1 answerMay 28, 2021Our lab intends to purchase plasmid kits but we do not know if we need any requirements or laboratory accreditation to work with them. Relevant answerMichael J. BenedikMay 28, 2021AnswerEvery country is going to have its own rules so you need to check locally. I would guess there is a bio safety office at your institution. But in general if the plasmid is in normal strains of E. coli and not carrying any toxic or potentially dangerous genes then you usually do not require any special precautions or accreditation beyond what you might need to do standard microbiology. View0 Recommendations

Anna Clouvelasked a question related to PlasmidsCan the BRAINBOW technique be applied to human epithelial cancer cell lines?Question2 answersMay 25, 2021Hello, For my current project I m looking to transfect a human cancer cell line, more precisely the A431 carcinoma cells, using a BRAINBOW technique. The aim in this is to be able to differentiate individual cells when looking at multicellular migration and cell contractility. I did some research, and found that BRAINBOW 3.2 had been used on a human neuronal cell line, as most of the research so far has been done on mice. This is my first time working with something like this; would it be possible to transfect the human cell line using a plasmid, so that they would all express different colours when they form a monolayer?Any advice is welcome!Relevant answerAref WazwazMay 27, 2021AnswerHi. I hope the following website could help: Recommendations1234567891011… 32AdvertisementJoin ResearchGate to find the people and research you need to help your work.20+ million members135+ million publications700k+ research projectsJoin for freeSimilar topicsPCRCloningPlasmid CloningBacterial TransformationGel ElectrophoresisPrimerMolecular Biological TechniquesReal-Time PCRCell CultureAntibiotic ResistanceHighly-cited researchers

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