Need suggestions for degenerate primers
Posted 10 February 2002 - 10:48 PM
I choosed the most conserved seq. and change it into nucleotides. I got 2 degenerat primers now. but I can't amplify it even I try to find the conditions for PCR.
Maybe I have wrong annealing temp. I'm new for this and I don't know how to solve this. Any suggestion? How to find the perfect conditions (annealing temp.,Mg++ etc.) for my degenerate primers?
Posted 13 February 2002 - 09:45 PM
If you can get access to a temperature gradient machine, set an annealing gradient to range from 10 degrees below optimum annealing to 10 degrees above optimum annealing. This should help to achieve an optimum annealing temp.
For other factors such as MgCl2 and template/primer concentrations, its a matter of trial and error.
Posted 18 November 2002 - 05:24 AM
If you couldn't see any PCR product, try nested PCR.