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Recombination with pHELLSGATE12 vector

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#1 resmi



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Posted 29 October 2010 - 01:24 AM


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)which was very near to region of recombination, 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.........

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