Hi, plz help me, I have cloned a gene of 3.5kbp, using already cloned plasmid as template, I just added 2 restriction sites and stop codon in the primers. ,in a vector of size 8.9 kbp, I have screened positive colony by plasmid isolation and further by single and double digestion. getting an accurate size of 12.5kbp and (8.9 and 3.5) respectively. But after sequencing 9using same primers) initial results are giving match with E.coli genome instead of with my gene ( rat gene). I tried with internal primers also, but they are not working. My primers are very specific. there are no chances of recombination bec. I am getting right size and PCR band after performing PCR
hello, you could sequence your PCR product with the primers you used to produce it. that will tell you if you are amplifying the right thing and check you template maybe thats not the right thing.
Hi,
It looks like whatever primer you are using to sequence is going in the wrong direction :-). Most of cloning vectors contain conventional sequence as SP6, T7, T3, M13fw or M13R that can be used for sequencing. They are very convenient to use, because most sequencing companies have already the primers in stock and sequencing reaction already set up. So check your vector map and use a couple of them. Another extra step you can do is to check your colonies with enzymes for restriction sites other than the ones you added.
@Dear Katrin : I am giving same primers which I have used for amplyfying my gene , I am checking my template on gel after restrction digestion I get 3.5kbp bands(gene szie) , I wil sequnce the template again .
Dear Elisa Garcia: My gene is in Lenti viral vector under promoter EF1 alpha promoter. I have used that primr also and got same result. First I cloned my gene in cloning vector pjet, I gave T7 primer and getting same result. yes I have procured enzymes to digest internal restrction sites . But I am wondering how come isnpite of primes being specfic , I am getitng results like this? with exact gene size. Thank you v much for the suggestion
Lenti viral backbone has lot of repeating elements in it. Make sure you are doing your subcloning in Stbl2 or Stbl3 strain of E.Coli that is specifically made for propagation of viral sequences. If you are doing in any other strain apart from these you will surely have recombination events. Hope that helps.
@Nilam Sinha , Dear Nilam,, thank you , I tried subcloning in STBl2 but due to endotoxin contamination and degradation of plasmid bec of it, I shifted to NEBstable https://www.neb.com/products/c3040-neb-stable-competent-e-coli-high-efficiency , purchased form NEB, this strain has high effiency and enda negative . I have checked the size and size are accurate (12.5kbp - vectoe 8.9+3.5gene)
I think it's not unbelievable to retrieve a bacterial DNA fragment with the same size and the same restriction pattern than you rat gene. It's difficult to believe but it can happen.
Maybe you cloned a chimeric fragment of your rat gene with a bacterial DNA fragment. During polymerisation, Taq can shift from a DNA fragment to another strand. Because of this property, studying alleles in polyploid organisms can be a nigthmare...
I think you should be sure to work with "genomic DNA-free" plasmid samples (i) to perform your first PCR and (ii) to sequence your recombinant clones. Instead "quick and dirty" plasmid extraction protocols/kits, you should spend few more hours to perform more careful protocols (eg. careful alcaline lysis, RNAse, Phenol/chloroform, Ethanol and PEG-MgCl2 precipitation, etc. It's not more expensive, just longer and you can get cleaner sample than with column-based protocols (data not shown ^_^)).The cleaner your DNA is, the easier your subsequent reactions will be.
When performing your first PCR, don't use too much DNA template nor too much Taq enzyme. The more is not the best, just more artifact and less specificity.
I think you should use more quality control steps during your cloning process : you said you already have another specific primer set targeting your gene, you should use them during your screening step before performing sequencing steps : restriction pattern + PCR screening (in the order that suits you : PCR screening is more expensive but quicker and maybe more stringent).
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