ligation troubles... - (Jul/14/2004 )
Vector: salI/nheI, insert is 39bp long and same digestion terminus, neb T4 dna ligase. I try different insert:vector ratio, ligation condition, but....NOTHING!
Have you got any idea?
I'm totally new to this and I've no scientific backing for it but I did read someone's post that you should use less than 10ng DNA/ul of rxn. I did try a rxn where I used more than 10ng/ul and another one with less than 10ng/ul and there was a marked improvement (more upshift) in the one less than 10ng/ul.
How did you get your 39 bp insert, PCR product or cut from a vector?
A common problem with insert digested from PCR product is the efficiency of digestion. Sometimes the digestion simply doesn't work even you have enough extra bases outside the restriction site. In this case, first do a TA cloning, then cut the insert from the vector and subclone it.