Ideas wanted-How to clone 600bp gene with two 5' 55base overhangs? - (Mar/20/2014 )

Hi

I have a 600bp PCR prduct but I need to add 55 base single-stranded overhangs to both 5' ends.

Any ideas?

Many thanks

Do you need precise ssDNA regions? The easiest way to do that would probably be with the NEB USER enzymes. Make a PCR primer having your 5' 55 bp, a single U, and 20 bp matching your existing PCR product. Do PCR with this, using Taq (high fidelity enzymes will not incorporate U, except for a version sold by Agilent). Then digest with USER enzyme, which will cleave the strand containing the U. This will leave a 55 bp 3' overhang, which may not be what you want.

If you need a 5' overhang, you probably have to ligate adapters. The adapters must have at least a short region of dsDNA - 12-16 bp will work. Best would be to add these with a cohesive end ligation, perhaps using an offset cutter, if you need exact sequence.