Does a single colony of E.coli contain more than one plasmid
Posted 27 October 2010 - 08:53 PM
I am working on pHELLSGATE12 vector for RNAi in plants. Initially I clone my gene in pDONR221 vector and LR recombined into pHELLSGATE12 vector. I got numerous colonies on spectinomycin plate which on colony PCR and sequencing confirms the presence of insert (Using gene specific primer I got gene preceeding some regions of (approximately 300 bp) pDONR221?????). But on sequencing with pHELLSGATE specific primer (CaMV and OCS) I got approximately 800 bp reading in both orientation which is of the pEHLLSGATE vector region only. So whats happening. I was confused???????? Does anybody know the solution......... If a single colony contain more than one plasmid how should I proceed...
Posted 27 October 2010 - 09:02 PM
It would be nice if you could confirm that the sequencing primer site was close to the actual insert (less than 800bp). As far as I know, sequencing reads are usually 800 bp and if you primer site is too far, you will just get the vector sequence and no insert sequence (even though the insert is present). I did not quite understand your 'more than one plasmid question'. Do you think that you have a colony with a realigned vector and a vector with the insert???? Kindly clarify.
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Posted 29 October 2010 - 01:16 AM
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Posted 16 December 2010 - 11:53 PM
Did you find the answer?? I have a question.
Is your HG12 is called pStargate?? If so there might be a problem with your construct.
Try to match/blast the sequence result against your vector sequence and identify where it is amplifying. You will get an idea.
In my experience, the CSIRO RNAi vectors has some kind of problems like intron reversal etc.