Degenerate PCR troubleshooting - (Aug/16/2005 )

I am attempting degenerate PCR, I have checked my template, it is good. I have increased my primer amounts to 50pmol as I was recommended, and I am using touchdown PCR, with 16 cycles were the temp drops sequentially. I am now trying different amounts of template. What amount of template do people generally recommend, how big a fragment would people try and amplify by degenerate PCR, is there a limit (what I am trying is quite large, nearly 1kb), and does anybody have any other recommendations for things I could vary that have previously given them rewarding results?

I've used touchdown as my standard method of amplification for a couple years. I routinely use 1-5 ng of template for a plasmid, though I never bothered to quantitate genomic DNA (just added 1 ul of whatever stock I had on hand into a 100 ul reaction). My fragments have been 1.2 kb in size, and I never had a problem with them. For larger fragments, just increase the elongation time (suggested time is 1 min/kb, I usually double that just to be on the safe side).

Just play around with your Mg concentration. Also, do you have no product at all, or just aspecific product, or aspecific product as well as specific? How big are the degeneracies, and where are the degeneracies to be found?

I am getting no product at all, not even a non-specific smear. Template has been tested and is good. Am using cDNA template, was using 20ng per rxn.

These are my primers, seemed easier than trying to explain how degenerate they are. I will try playing with my magnesium concentrations next.

CiAAt/ca/cGiAAa/gTAt/cTTt/cTGGCGiAT

AiCCt/cTTa/gTTa/gTTa/gCAiGTt/cTGg/aCA

What range of Mg concentrations would you try? I am using PCR rxn ready mix, so it will require adding additional Mg to this, can't really decrease the concentration therefore.

If you're getting no product formation at all, just lower your annealing temperature, go 3-5 °C below the lowest of your touchdown.

Vary your Mg between 1 (if possible) and 5 (this is very high, I know).

If you then still see no product, try to check your primers.

Try doing a PCR where you use one degenerate primer and another one that has been tested to work on your cDNA.

Between 1 and 5 what? pmols? (for Mg concentration)

mM (millimolar).

thanks for the clarification.

I altered the MgCl concentration as much as I could, and dropped temp, did get a low Mw smear at highest MgCl2 conc. I thought I would use this product in a second round of PCR, any other suggestions?