Problem about plasmid constrution - (Aug/03/2007 )

After many times PCR, I can not the sequence I needed. So I want to use two restriction enzymes to cut one DNA sequence from plasmid one (about 100ng), and after that I make DNA gel. If I can use DNA extration from gel kit to get the DNA sequence, and if there are so little DNA left after the kit that I can not construt another plasmid.

If the starting DNA is 100ngs for digestion, you might lose most of it in the columns when you purify it. Try to use more of the PCR product or make more PCR preps to increase the starting material.

I agree, with scolix's acessment. You do need more DNA. I would cut 10x more DNA. (I tend to be wasteful...but better to waste reagents then contantly worry about losing DNA.)