Troubleshooting degenerate PCR - what is too degenerate (and other Qs) (Oct/19/2006 )
I've been trying to optimize several PCR reactions involving degenerate primers and have some questions:
1) what would you consider too degnerate to bother with? I've been lucky enough to find some primers that are as low as 12-fold degenerate, some that are 2,000+ fold and some inbetween. What's the cut-off? When should I scrap a primer and not even bother ordering it?
2) MgCl2 gradients - I've been running gradients from about 1.5 to 4.0 mM final concentration. Is that reasonable? Should I be going lower? Higher?
3) annealing temps - I haven't been paying too much attention to the calculated annealing temps of my primers (since they're dengenerate the annealing calculated temps probably aren't too great anyway) but instead have been doing a gradient from about 46-64*C. Is there a general guideline about how low is too low? Yes, I want PCR products but it seems if I go low enough I'll just get a bunch of junk. Does anyone have any experience about how low this is?
4) touch downs - I gave these a shot (starting at 64*C and decreasing by .5*C/cycle for 35-40 cycles) but didn't have much luck. Has anyone successfully used touch-down PCR and if so, is there anything obvious I'm missing?
5) BSA include it or not? What's a good concentration to use? I've been using a final concentration of .32mg/mL. Not sure why, basically I was told to try using it but no one could give me a good idea of what the final concentration should be.
.... and yes, someone did make of with my Maniatas/Sambrook and no, I don't have cash for a new set.
I feel the most important part is the primers you design. I use the CODEHOP program, do a pub med search to look it up. It helps to design really good primers.
the 3' end of the primer is the most important. If you have good binding on the last 8 bp or so, then many other sins can be forgiven. 2000 way is a bit high, but not completely out of the question. I would say you have good reason for optimism. You might want quite a bit more primer than usual, since only the matching ones will prime.
I find PCR with annealing temperatures below 50 to be pretty ineffective. Depending on the GC composition, it might be important to add 2-8% betaine or DMSO (I think betaine works better). I would use Taq or a Taq + proofreading enzyme mixture, rather than one of those fancy enzymes.