Molecular Cloning

Molecular Cloning

  • Closed account added an answer:
    I want to combine CRISPR and AAV mediated gene targeting and wonder whether somebody has tried it or has information on critical steps?
    .

    Dear Ortiz,

    Thank you for the clarification. In fact, I have made that point very clear earlier in this post. rAAV for single stranded DNA donor template delivery, NOT to deliver gRNA or Cas9.

    Manuel

  • Christian Riesenfeld added an answer:
    Can anyone tell me a website to find the names of bacterial genes, their types, and their functions?
    For example, EXDO-D and EXDO-K2. What are their actual names, functions, and the differences between them?
    Christian Riesenfeld · University of West Florida

    definitely visit  http://www.ncbi.nlm.nih.gov/COG/

    Lucia's answer is great as well 

  • Bilge Ercan added an answer:
    What is the general procedure for a yeast cDNA order?

    I want to express yeast specific protein in E.coli. My design is to order cDNA and I want to amplify the gene of interest using PCR. I did not order cDNA before so I have no idea which things I should pay attention. I'd be very glad if you can provide some information.

    Thanks all! I got the genomic DNA!

  • Urda Rüdrich added an answer:
    How do you integrate one resistance cassette from a plasmid to another?

    I am a really beginner in knockout generation and mutagenesis and it is my first trial to generate a double knockout mutant via heterogramic conjugation. The problem I have is that I need a second resistance cassette for the second KO gene, which is not on the standard plasmid we use for cloning (pCR 2.1 TOPO). How can I obtain the resistance gene from another plasmid and bring it into the topo vector? I think I need the plasmid sequences and restriction sites but where can I find it? 

    Urda Rüdrich · Hannover Medical School

    Maybe this also helps...

  • Nirmala Arul Rayan added an answer:
    Why are my bioanalyzer results are very different from agarose gel results?

    I'm currently trying to prepare DNA libraries for Chip-seq, the problem is I have ran some IP'ed DNA samples from the ChIP experiment on the high sensitivity bioanalyser chip and on a 1% EtBr gel the results are very different.

    The 3 sample lanes in the gel corresponds to  sample 2,3 and 8 (1st image) on the bioanalyzer PDF (see pdf)


    I have also tried the library prep which also shows the correct result on a agarose gel(last image).

    The library prep was prepared using the NEBNext illumina Prep kit with 70ng (with size selection) and 5ng (no size selection) of DNA from sample 3.


    Does anyone have experience with this?

    Nirmala Arul Rayan · Agency for Science, Technology and Research (A*STAR)

    Hi,
    If this is not DNA from library prep but IP DNA; the discrepancy you see between the agarose gel and high sens bionalyzer results is because of sensitivity differences. Try running your sample on DNA 1000 chip. Then the two results could be comparable.

    Alternatively you could dilute your samples before running on a high sensitivity chip, if all you want to know is the size distribution.
    All the best

  • Yaghoub Safdari added an answer:
    Does anyone have an idea as to why I am unable to get the correct size of human protein cloned in a pET28a vector?
    I have cloned a human gene into the pET28a His tag vector but I am unable get the correct size of my protein. I have confirmed recombinance by sequencing and restriction digestion.The correct size of my protein is 55 KDa but I always get a band of 75 KDa. Please give me suggestion on how to get the correct band?
    Yaghoub Safdari · Golestan University of Medical Sciences

    I believe that neither His-tag nor post- translation modifications (Glycosylation and ect.) are responsible for the difference (20KD) you observed.

     From the first ATG codon, which exists on the plasmid map before the first His-tag, to the stop codon, which lies after the second His –tag, will potentially lead to a molecular weight of only 6.8 KD ( approximately 1/3 of the difference you observed).

    Despite their several advantages in producing recombinant proteins, bacteria are unable to accomplish the required post-translational modifications.

  • Jonathan D Finn added an answer:
    Can someone provide a complete clone of humanFANCA gene ?

    Our lab is Interested in Function Study of  FANCA protein in Wnt signaling for that we want to over express FANCA in mammalian cell line .

    Regards

    Jonathan D Finn · Arthrogen

    You could always use RT and a specific primer set to amplify the cDNA from expressing cells. According to GeneCards, it is expressed in 293 cells or you could probably amplify it from a human blood sample. 

  • Carl Angelo Medriano added an answer:
    How do you troubleshoot failed transformation of plasmid to host competent cells?

    We received a plasmid delivered by mail and performed transformation experiment following protocol. the plasmid is used is topo with 3.9kbp. we used AK plates for blue/white screening due to the ampicillin and kanamycin resistance. After selection of white plates, innoculation and plasmid extraction was performed. Clones' plasmid were checked for band using pcr electrophoresis. It is consistent that all clones produced bands way larger than the target gene band.

    We used gene specific primers 

    Carl Angelo Medriano · Korea University

    thank you all for your comments. Actually whatI did is perform PCR of the plasmid received before transforming to the host.

    We were able to confirm the target before transformation but the clones all have way longer bands as compared to the pcr product before transformation. Also with the presence of two antibiotics where the plasmid is resistant to, we also thought that the those growing would most likely containing the plasmid.

  • Kaushal Kumar Bhati added an answer:
    How can I identify know intron and exon regions in a given nucleotide sequence? How does a SignalP work?
    Suppose I have sequenced one of my clones, how can I know which regions constitute the exon, intron, splice sites, splice variants, signal peptides and disufide bridges? What is the easiest method for getting the translated nucleotide sequence, and how to predict the secondary structure of the translated protein sequence in groups? I also want to know how SignalP software works.
    Kaushal Kumar Bhati · National Agri-Food Biotechnology Institute

    FGENESH is server based tool...u dont need any licence for that....though daily use and runs for this are limited......

    sory for late reply.......i did'nt get any update...from this post...if in case u need further help...msg me...

  • Dylan Levac added an answer:
    How do I clone qPCR product with sybr green to T-easy vector?

    I am trying to clone qPCR product with sybr green to T-easy vector even after purification and adding A at the ends, anybody can help?

    Dylan Levac · Mount Allison University

    Hi Peijin,

    I would first verify that your ligase operates at 4oC.  That might be your problem.  In my experience I have never run a ligation at 4oC.  Most commercially available enzymes suggest working between 12-16oC for overnight ligations.  Invitrogen's T4 DNA ligase, for example, suggests 16oC for 24 hours if you're trying to ligate blunt ended fragments.

    Oddly enough I ran in to something exactly like what you're hypothesizing last summer while trying to clone some genes.  It was Safe-T-Stain from Bioshop - an ethidium bromide alternative. I showed in my troubleshooting that this product, Safe-T-Stain, completely inhibited ligation reactions as well as E. coli transformation.  The first was shown by trying to amplify across the multiple cloning site, the second shown by transforming uncut vector.  Both experiments were performed with DNA which had been gel purified from gels containing Safe-T-Stain, or SYBR Safe DNA stains. SYBR Safe treated DNA worked fine, Safe-T-Stain treated DNA couldn't be ligated in to vectors, and uncut vectors couldn't be transformed in to E. coli.


    If you think your problem is associated with a component of the qPCR master mix, then I would run a standard, non-qPCR reaction, harvest the amplicon by gel purification and try to clone that.  Be sure to use a non-Taq enzyme for the purification, then A-Tail etc.  That way you're as close to your qPCR conditions as possible.

    Best of luck. 

  • Moumita Sarkar added an answer:
    What is the molecular weight of mTOR expressed through pcDNA3/AU1-plasmid?

    What is the molecular weight of mTOR expressed through pcDNA3/AU1-plasmid containing rat mTOR gene. I am using this plasmid and when I immunoprecipitate myc tagged mTOR with myc antibodies, I am getting a band at around 100 kDa mark. The m.wt of human mTOR is around 250 kDa.

    Moumita Sarkar · Indian Institute of Chemical Biology

    Hi.....that might be a non-specific band. You should check it in your input lanes. You should get it at 250.  I agree, u should check first the expression of the gene by western blot. You might need to check transfection as well as your immunoprecipitation procedure.

  • Indra Kurniawan Saputra added an answer:
    Does anyone know what factor makes re-circular plasmids after restriction and ligation in the cloning method?
    see above
    Indra Kurniawan Saputra · Bogor Agricultural University

    hi ajay...

    thanks anyway

  • Yuan-Yeu Yau added an answer:
    How can I confirm Agrobacterium (GV3101) transformation?
    I am facing problems in confirmation of transformed GV3103. Transformed bacteria is able to grow on antibiotic selections media (Rif+Gent+Kan) while the wild type was unable to grow. But colony PCR and PCR on mini preps were negative. What can be the problem? I tried different concentrations but still the bacteria was able to grow.
    Yuan-Yeu Yau · Northeastern State University

    Hi Zeesham,

    The rearrangement of transgene cassette on binary vector may cause problem too.

    1. The rearrangement (such as deletion) might have occurred and deleted your gene cassette in Agrobacterium. For example, if you have homologous DNA sequences or repeats (especially the long repeats) which flank your gene-of-interest (GOI) (for example). After this binary vector was transferred into Agrobacterium, there is a chance that this GOI be looped out through homologous recombination. If this is a case, then: (1) when you do PCR (assuming you are PCRing GOI), the 'original' cloned plasmid will give you a band, but the mini-prep DNA from Agrobacterium will not generate a band.  (2) Even your GOI is deleted, the Agrobacterium still harbor the 'leftover' binary vector backbones and the backbones can still replicate In the Agrobacteria (with kan resistant). Therefore, the Agrobacteria with these leftover backbone can still happily grow on medium with Rif + Gent and Kan. 

    2. That is the reason why instruction sheets from some companies producing competent cells (especially E. Coli) will tell us the genotypes of the particular bacteria they have used in the kit. One of the important genotypes is 'recA', a mutated RecA. With recA, the risk of our plasmids been modified (rearrangement through recombination) in those cells will be reduced. 

    3.Except this RecA gene, there are other mechanisms involved in rearrangement of  T-DNA. Try to avoid using too many long homologous DNA in the construct.  I attached a paper for your reference.

    Good luck on your research,

  • Terrens Saaki added an answer:
    Does someone have a protocol for home made Gibson assembly master mix?
    Gibson assembly is supposed to be seamless in cloning especially when you want to make a construct from different pieces (more than 2). Since the commercial kit from NEB is expensive, I would like to have my own home made kit.
    Terrens Saaki · University of Amsterdam

    Hi @Christian Cantos,

    I'm using the protocol from Miller's lab! See above. And it works nicely. Make sure you do the DpnI treatment this is to digest any wild type plasmid in your reaction.

    Briefly:

    I PCR amplify every fragment (20 bp overlap hangs on inserts only, this way I can use the same primers for the vector every time I need it), 1 hr DpnI (NEB) treatment (0.5µL in 30 µL reaction) at 37 degrees, column clean up all fragment. While DpnI reaction is taking place I run 5µL on gel to see whether PCR products with expected sizes are amplified. I use 2X of each inserts! And this works for me! Also take along a control by mixing 15µ water with same amount of DNA material as in your GA mix. Transfer both in TB10 ( I think any other strain could be used) high competent homemade cells. You need high competent cells for this to work.

    Cheers

    TS.

  • Faezeh Kharazyan added an answer:
    Has anybody used PCR product from agarose gel band for cloning DNA?
    After electrophoretic migration of my PCR products, I cut my gel band. Then, I have purified my DNA using the Qiaquick gel extraction kit. After nanodrop, I obtained a correct DNA yield (about 20ng/ul ), but I had also an absorption pick at 230nm (A260/230=0.02). I don't know what is it. Agarose or co-purified molecule from the kit buffer?
    I'm confused because I want to use these purified products for cloning. Is someone experinced similar problem? Do you think that the cloning may work with abosrbance ratio?
  • Jose MARIA Buesa Galiano added an answer:
    Does anyone know an iInsoluble binary protein complex?
    Does anyone know a protein complex that is only soluble in yeast (or any other eukarytic cell) and insoluble in E.coli? Id appreciate if anyone could share some examples.
    Jose MARIA Buesa Galiano · CBGP centro de biotecnología y genómica de plantas

    Hola Arturo, The PI3Ks complex (Phosphatidil inositol 3 kinases) with two subunits  and 120, 50 aprox Kd of molecular weight( for alpha form), fail to be expressed in E.coli and has to be cloned in insect cell to get a soluble active form to assay . I hope this aid to you. Buena suerte

  • Sanjay Mishra added an answer:
    Can someone please explain why there are 2-3 bands of my protein after purification?

    I realized that this often happens only when I perform purification. My protein is fused to a 60kDa tag and my protein it self is ~20kDa. So taken together, the fusion protein should result in ~80kDa band. When I express this protein, it is a single band. However, when I purify the same protein on the Nickel packed column, more than 1 band (bands below the red arrow) is eluted out. What could be a plausible reason for this?

    I hate attached the purification image here.

    Sanjay Mishra · IFTM University

    In my opinion, it is very common in this context. What exactly, you are advised to work on with probing with specific antibody followed by 'Western Blotting' technique. you may probably obtain the good results likely to be reproducible. further queries are most welcome.

  • Carlos G. Acevedo-Rocha added an answer:
    Does anyone have a troubleshooting guide for CPEC reaction?
    Problem with CPEC reaction.
    Carlos G. Acevedo-Rocha · Max Planck Institute for Coal Research

    Do you mean Circular Polymerase Extension Cloning? You should check this: http://www.nature.com/nprot/journal/v6/n2/abs/nprot.2010.181.html 

  • Nadiia M Lypova added an answer:
    Does anybody have an experience with pop2136 cells and vector pEX2?
    It seems I have problem with the plasmid stability.
    Nadiia M Lypova · Innovation-manufacturing company "Biowell"

    Dear Joannie Kamens,

    thank you for your reply, I keep the plasmid in XL1-Blue too, but I have problems during the induction - some of clones are without any traces of target protein after induction. I can explain it by the problems with plasmid stability during the cultivation of bacteria...

    Do you have any experience in induction of protein?

    thanks

  • Sagar Krishna Bhat added an answer:
    Why did LIC for my full length gene and constructs not work?

    Hi all I have a full length (FL) sequence gene and few constructs of the gene  to produce a functional protein.. which I would purify if all goes well.

    Firstly, I did normal cloning of my FL gene and its constructs i.e. Molecular cloning of them using Pcold vector  TF and Prs2.. It seemed positive till now  and I have send the clones that were positive  for sanger sequencing (did mini prep etc for the same using Kit) awaiting results.

    Now the second part which is that I tried LIC and ran gels I obtained faint bands  which was of the primers and not the desired sequence.

    Tried with optimizing the PCR by adding DMSO.

    Please suggest how could solve this problem.

    Regards,

    Ratish Raman

    Sagar Krishna Bhat · Kaypeeyes Biotech Private Limited, Mysore

    I generally work with PCR enchancers from Qiagen or Invitrogen, as it is less of a hassle. Check if the primers annealing temperature experimentally, because it  can be lower than what you have calculated. Also check for if there are serine, leucine or arginine residues in the sequence you are amplifying, if you do, then you will have to redesign your primers.

  • Dylan Levac added an answer:
    Can anyone help with a problem regarding Taq polymerase for a 1500bp tempalte?

    My template amplifies when I use Hi fidelity kapa and not when I use Taq polymerase   (normal/regular) . Is there any limitation to template size for taq polymerase? I had given it 1 minute and 45 seconds for extension and it still did not amplify. 

     

    Dylan Levac · Mount Allison University

    If you can amplify from this template using a HF polymerase, I would determine the true extension time of your Taq.  You can do this by amplifying a known gene of a purified plasmid template.  Use progressively shorter extension times until you can't observe a strong amplicon.  

    In my experience Taq from different commercial sources have dramatically different extension times.  Takara ExTaq can generally do 1 Kb in 30 seconds, even though they suggest 1 min (I believe?)  where as Fermentas or BioShop Taq takes around 90 seconds per Kb.  Personally, I don't even use those enzymes to A-tail for cloning.  

  • Gul Imriz added an answer:
    Does anyone have any knowledge about Chitin azure?
    I could not find any method to prepare chitin azure. Before, Sigma was providing crab shell chitin for chitinase activity but now they have discontinued that product? I have to perform an in vitro test for chitinase activity.
    Gul Imriz · Bahri Dağdaş Int. Agric. Res. Inst.

    Dear Awan

    I am sorry if i am too late to answer. There is a paper about chitin collodial. You can see the paper name below. I hope it will be helpful. The paper is;

    MURTHY N. and Bleakley B., Simplified method of preparing colloidal chitin used for screening of chitinase-producing microorganisms, Current Microbiology, 57(5), 503-507, (2008).

  • Prashant Kumar added an answer:
    What is the reason for the difference in protein expression level?

    I am currently having two truncated gene  from the full length gene to produce a functional protein. both of the gene having the same sequence, unless the first version having a longer amino acid code (30 amino acids, which 30% of those coded for Gluthamine). I cloned both gene into pET-28a and with the cloning host BL21. 

    However the result were, the first gene which coded more amino acids, was overexpressed with a very thick blot of the SDS-PAGE gel. however for the second gene which is shorter was not overexpressed very well in the gel. 

    thanks.

    Prashant Kumar · Indian Institute of Technology Madras

    Hi

    A smaller size protein are relatively less stable especially in  a heterologous system. To overcome this problem, you can tag your proteins with glutathione-s-transferase, maltose binding system, chitin binding protein. The tag can be removed by either site specific protease or chemically. 

    All the best.

  • Thomas Dobrenel added an answer:
    My BP reaction does not work in the case of multigateway cloning for the non-attB1-attB2 cassettes. Can anyone lend their expertise?
    I am creating a multigateway vector with the cassettes attB4-attB1R, attB1-attB2 and attB2R-attB3 and I have no problem for the B1-B2 but I fail in getting any colony for the two others. I've been using the B1-B2 as a control in every reaction I did and so far, it worked perfectly each time. I've used different strains and stocks of bacteria, played with the ratio vector/PCR product, re-ordered a fresh BP Clonase II Enzyme Mix and still, it does not work... Can anyone provide me some ideas of things to try?
    Thomas Dobrenel · Umeå Plant Science Centre

    Hello,

    First of all, I would like to thank you for your reply. With a colleague, we already tried to play with the PCR product concentration and the ratio vector : PCR product and we have also tried to increase the incubation time up to 24hrs but so far, it didn't help.

    I have sequenced the donor vectors and I found one mismatch of one base in the attP site but it seems to be far away from the recognition site by the enzyme or at least not in the exact recombination site between the attB and the attP and I don't really beleive this may be the problem but I am very open to change my mind if someone tells me this is important. I anyway wanted to order some fresh vectors but I didn't find them apart but only included in VERY expensive kits.

    I've checked also the TOPO cloning between attL sites but the only vectors I've found were for attL1-attL2. Let me know if you already seen something similar for the other cassettes.

    As I am a bit afraid that the quality of my PCR product is not good as well, I am right now subcloning them in another vector to amplify them and sequence them to check the sequence of the attB borders and the idea afterward would be (if the sequence are OK) to linearize the vectors and try the BP between the DONR and this linearized PCR product-containing vector.

    I will keep this post up to date if I am successful with this strategy (so, if I don't post anything, this means that I've not been so lucky, unfortunately) and, please, don't hesitate to tell me if you see anything else I can play around or if you know anything about ordering fresh vectors for a reasonable price or TOPO cloning between attL4 and attL1R or attL2R and attL3.

    Once more, I thank you for your reply.

  • Alexandre Gonçalves added an answer:
    shRNA cloning- Why are my recombinant colonies taking a long time (2-3 days) to grow on ampicillin plate?
    Recently, I was cloning some shRNAs into Clontech`s pSIREN-retroQ vector (U6 promoter). After ligation, I transformed them into XL10 gold/SURE2 cells according to the manufacturer`s instruction. But when I grow them on an Ampicillin (100 ug/ml) plate, they are taking 2/3 days to grow. I was getting correct insert from those colonies though. But I got another problem, when I transfect the vectors into cell lines (MCF10A), they are not expressing which I confirmed through qPCR. What might be the reason behind it?
    Alexandre Gonçalves · Intellects - New York, USA

    Can you verify if you have any point mutations? If your colonies take 3 days to grow I think you may be looking at strange recombination events.

  • Andres Esteban Marcoleta added an answer:
    What is a good vector mapping software?
    Can anyone suggest a suitable vector mapping software? Suppose I have a sequence of a vector, I want to know the promoter, terminator, antibiotic resistance etc. in a region. I used NEB cutter, but this one is useful only to track restriction sites and CDS.
    Andres Esteban Marcoleta · University of Chile

    I tried APE (MacVersion) and worked wonderful! I recommend it. There is also a Windows version.

  • Grant Gallagher added an answer:
    Why are there multiple missense mutations in my cloned gene?
    I am working on cloning a 3 kb gene into pET-47b using engineered restriction sites. Sequencing showed that my gene was successfully ligated into pET47 but it contains 7 missense mutations. I sequenced a plasmid from a 2nd colony and it again had 7 missense mutations but they were at different locations. I am new to cloning and I was wondering if this is a common problem? So far I have 3 guesses of what the problem could be:
    1) UV light while gel purifying the PCR product digest (I think I tried to keep the UV exposure to a minimum. I have also tried not directly exposing my desired lane to UV in the past, instead I used a small amount in a separate lane which I can use to determine the position of the band. I do not remember what method I used for this prep).
    2) Old/bad polymerase used in the PCR amplification. I am using the KAPA2G Robust kit which I understand to have very high fidelity but it is close to 2 years old.
    3) Would a high concentration of dNTPs in the PCR reaction cause missense mutations? I did increase the concentration of dNTPs in the PCR reaction (used 1 uL) because my gene is 3 kb.
    Are any of these problems a likely culprit?
    Grant Gallagher · HUMIGEN - The Institute for Genetic Immunology

    Great News! THanks for updating us. GOod luck with the rest of your work

    best

    G

  • Joseph Ndika added an answer:
    Does anyone have experience with no PCR product during site directed mutagenesis?
    I have been trying to incorporate two point mutations in a 8kb plasmid using the Quick Change II site directed mutagenesis from Stratagene. The two mutated sites are 8bp apart. Also, I used the Stratagene primer designing software to design the primers. So far I have been unsuccessful getting a band after PCR. Couple of times after DpnI digestion, I did bacterial transformations (even though there was no band in gel), however, did not see any colony. Moreover, using the same PCR set up, I see blue colonies on X-GAL, IPTG coated plates for the control plasmid that comes along with the kit. I pretty much follow the kit guideline to set up the PCR reaction mix. Also, I tried with different amt. of template (5ng-50ng) keeping the primer concentrations constant, but no luck yet! Every time in gel I see a strong band for primer-dimer. Bottom is the PCR set up I used. Any idea will be highly appreciated.
    Initial denaturation: 95C for 3min
    18 cycles of 95C for 50sec.
    55C for 50sec.
    68C for 18min.
    Final extension: 68C for 8 min.
    Hold:4C.

    I always run a gel taking 10ul. of the reaction mix after PCR and use the rest for DpnI digestion.
  • Mai-Britt Vadekrog Jensen added an answer:
    Does anyone have experience or information regarding difficulties in sub-cloning eGFP?

    I've tried five times now, using PCR to amplify and add 4 different combinations of restriction sites to try to insert into a pCDNA3 derivative. I've had great success with the vector with other inserts during this time but get no GFP inserted. Any suggestions? All I can think of is some structure in the GFP DNA that inhibits ligations. Perhaps switching to a different fluorescent protein is necessary.

    Mai-Britt Vadekrog Jensen · The University of Edinburgh

    I had some problems with subcloning GFP into TOPO TA vectors last year. A colleague advised me to add glucose to plates (I used 0.5% final conc.) and subsequently glucose in the overnight cultures (0.2% final conc.) prior to miniprepping. It worked a treat for me.

    Good luck

    Maia

About Molecular Cloning

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