I have a problem in ligation of 3kb PCR product(5'BamHI and 3' Pst1) into T-vector.
In ligation process, I use 1:3 or 1:5 ratio of T vector and my insert.
Ligation reaction at 4 oC for overnight and also try 16C for O/N.Moreover,I also try 22C for 2 hours and 16C for 3 hours also.
I use Ecoli,competent cell and incubate for about 14 hrs.I found white and blue colonies but the problem is when I run PCR I can't see any bands..
Could anyone give me some suggestions?
Hi,
Thank for your reply.
I used ex-taq which produce product with A-overhangs.
I don't do restrict my PCR product.I mean when I design primer sequence,I put restriction enzyme seq. at both site.
Do you mean you do plamid preparation first and do restriction analysis?If so,how can I choose colonies for plasmid prep?
Hi
3Kb gene is long size of DNA therefore it might be possible that PCR product from the super coil structure. after amplification PCR product eluted PCR product from Agrose gell and before ligation heat the PCR product at 70*C for 5min and keep immediately on ice for 5 min which lead to open the supercoiled DNA after that ligate your PCR product in 3:1 for overnight and then
transfom in E.coli
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