Help: PCR clone problem - (Mar/16/2008 )
I am doing a PCR clone, initially , I did PCR using Taq, and the sequencing result showed a extra T at the 5'-end, but no extra T at the 3'-end，My teacher told Taq is the criminal who add the extra T. so I switched to Fermentas Pfu DNA Polymerase, but this time, Invitrogen's Sequencing result show the same question? where goes wrong?
Is my Primer the criminal? should I buy a new primer?
thank you very much indeed:)
Sequencing results at the 3' end of PCR product sequences gives poor results, in general. Do not trust the sequence you get to reflect what is actually present on the molecule. If you want to see what is really there, you need to clone it into a vector, pick single colonies, and sequence the vector. Why do you care?
Thank You phage434 for coming to my rescue again
I will do PCR clone on in 2 days
I wish I could get a right plasmid