Making dsDNA from an ssDNA oligo - (Apr/18/2005 )
I've got some 66-mer single-stranded oligos which I would like to make double standed for digestion and ligation into a vector. Does anyone know of a reliable polymerase-based method to synthesise the missing strand? Unfortunately things are complicated by the appropriate primer to the 3' end of the oligo having a Tm of 38C.
Alternatively, are there other methods for synthesising the missing strand? Or will I just have to bite the bullet and order the complementary strand?
technically, you only need one primer if you still want to go the PCR route, so pick your better primer.
The primer with a Tm of 35C is the best that I can manage.
Are there any suggestions as to how to run the reaction? Specifically,
1. What sort of relative quantities of primer, template and enzyme should I use?
2. Can I get away with Taq or is something like Klenow necessary?
3. Should I try for just one round of extension, or a few cycles?
The experience of somebody who's done this sort of thing before would be invaluable.