For confirmation the cloning of my insert (shRNA) in pRNAi vector, PCR did not respond at all, but when I tested the extracted plasmids of the same colonies with restricted enzyme (scaI), I found positive colonies among them. I designed the forward primer on the shRNA (insert) & the reverse primer on the pRNAi vector.
Since you have a single restriction site it is possible that the positive clones that you have obtained might contain insert in reverse orientation.Due to this reverse orientation u might never get a PCR amplification but a positive insert release.
Thanks Krishnanand for your answer.
But I have two restriction site (BamHI & HindIII).So, why I do not get a PCR amplification?
Thanks Halle for clarifying me.In this case you could verify your positive clones by sequencing using vector specific primers which covers MCS site from both sides of insert DNA(this will tell u what u have actually cloned in the MCS site in your positive clones).
Further it would be helpful if you could answer these questions
Whats the expected size of your PCR product?
What do you see after the PCR reaction in gel? Do you see a smear,primer dimer or is it completely blank? If its completely blank try to run a positive control to make sure your PCR reagents are fine but if its not blank then you could try some other things too.
Hope these suggestions would help you.
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