ssDNA with Strandase Kit - (May/25/2007 )
I am trying to get ssDNA from my dsDNA PCR product using Novagen's Strandase Kit. This involves using a 5'-phosphorylated primer so that only one of the strands of DNA is chewed up. However, I am getting an incredibly low yield of ssDNA. I am measuring this through OD260 and I know that absorpance of ssDNA is less than that of dsDNA, but that doesn't account for a 10-fold decrease in product. Any help would be great. Thanks
Why do you need ssDNA?
You can get a linear amplification of ssDNA by using asymmetric PCR -- very low concentrations of one primer, and normal concentrations of a second. This will replicate the double stranded DNA each cycle, without making substantially more, and the result will be largely ssDNA starting with the primer in high concentration. This is probably a better way to get ssDNA than digestion. Another approach is to use the M13 origin still present in e.g. pUC19 along with an M13 helper phage.
I have tried asymmetric PCR before and it wasn't quite working out to get ssDNA like I wanted it to. It was probably because I'd never done that before and the conditions weren't ideal. But then we heard of the strandase kit that cleaves dsDNA into ssDNA if one of the strands had a 5' phosphorylated end.