Expression vector - (Mar/20/2006 )
Primers for kunitz trypsin inhibitor was selected from a paper and used for PCR amplification of cDNA extracted from plant sample. There was no distinct band hence selected the smear for elution and transformed into pTZ57R/T vector. Double restriction digestion could release desired 800bp size insert but sequencing did not show good result. I am trying to ligate it in expression vector pGEX and look for protein profile. Transformation in BL21 cells giving rise to colonies in ampicillin selection media but subculturing the same is not working. What will be the problem?
If you're looking clone a distinct fragment from your PCR, you're problem starts if you did not amplify this band. I'd look back at the PCR and troubleshoot and try to amplify the the 800bp band your looking for.
The smear you ligated could be anything.