I tried to ligate my insert (520bp) into TOPO vector (TA cloning).
My PCR product has concentration of 30~40 ng/mcl
I used the mixture as below:
PCR product 3 mcl
TOPO vector 0.3 mcl
Salt 1 mcl
D.W 5.7 mcl
Incubate at RT for 30 min
Than transfer the mixture into Competent cell.
However, I just got the blue colonies, without any white colony. I did try 3 times with the same results.
Previously, I did the same protocol for another construct (625bp), The results were good.
I don't know what could cause the problem for this new construct...
Please give my some suggestions.
Thank you so much!