Need suggestions for degenerate primers - (Feb/10/2002 )
I have to amplify 'ppk gene' from my isolated bacteria which I can't find the info from Genbank so I have to design primers by myself. I used the ppk gene information from Genbank by using amino acid seq. of many bacteria and aligned it with ClustalX and Genedoc.
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?
degenerate primers can be inefficient in the first rounds of amplification. To see if amplification will occur try reducing the annealing temperature to 10 degrees below what you are currently using, for the first 5-10 rounds and then set the temp back to normal for a further 25-30 cycles.
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.
I once did something like you're trying to do. I guess, you already check for the codon usage of that bacteria. The first reply was right. You should try to decrease annealing temp 5-10 degree lower than recommended temp from primer company.
If you couldn't see any PCR product, try nested PCR.