I have cloned a gene in pFastBac and confirmed its presence by restriction analysis. It was then transformed in DH10Bac cells carrying the bacmid. By site-specific transposition, the gene is to move from pFastBac to bacmid and this is confirmed by blue-white screening. I picked up the white colonies (carrying recombinant bacmid), restreaked them on another plate and those which displayed the white phenotype were subjected to bacmid purification. They were then screened by PCR for the presence of gene in bacmid using bacmid specific primers. I got PCR product corresponding to the size of the gene when I used gene specific primers and no PCR amplicons when I used bacmid specific primers. Does that mean that gene has not transposed into bacmid and still present in pFastBac even though the transformed colony was white?
Hi Ankita
You need to do some more experiments to conclude certain points.....why don't you try combination of gene-specific primer and a bacmid specific primer first......Some times one of the primers may be bad or there can be problem with PCR per se....you never know,,,,,,
check the possibilities one by one..
bye
I tried PCR with gene specific as well as bacmid specific primers but I didn't get any result but I guess I should try PCR with different conditions.
Hi Ankita
I think I was not clear above by 'combination of the primers' I mean use a single gene specific primer and one bacmid specific primer.....I suggested this because you mentioned that your gene specific primers are working fine.....sometimes the clones are also unstable over the time...and in later stages you may not abe able to get insert when bacmid specific primers are used.....so check this aspect carefully and also as mentioned by initially optimize well the PCR conditions....and most important...so see if everything is working fine put a positive control always using a fragement that always amplifies....
all the best
Was this solved Ajay?
If not, add 5% DMSO to your PCR reactions, use 52C annealing temp and prolong your extension time (with M13 primers). Are you doing colony PCR? Then add a colony to 50 uL water, heat it and disperse the solids, then use only 2ul as a template per 25ul PCR reaction. Negative background in transposed DH10Bac is a common issue and will be more of a problem if you have too much template DNA.
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