Ideas wanted-How to clone 600bp gene with two 5' 55base overhangs? - (Mar/20/2014 )
I have a 600bp PCR prduct but I need to add 55 base single-stranded overhangs to both 5' ends.
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.