Is it possible that if I have not included 6-8 bases near the RE site during primer designing, I can have trouble during ligation?
if the RE is not able to cut properly , then my insert and vector wont ligate well?
I am having trouble with my ligation.
I use 1:3 molar vector:insert ratio, 50 ng of vector in the system.
I also dephosphorylate the plasmid..
I have tried overnight ligation at 18degree C and rapid ligation at 22 degree C for an hour
if i transform only the plasmid i get colonies..
i also purify the pcr products
so my cells are OK
the only problem i see is that the ligation is not happening
is it faullty primer design??
am i stressing out too much and is it something simpler which is causing the problem ??