degenerate pcr - (May/17/2010 )
I'm quite new in laboratory works
It's my first time doing degenerate PCR
All I can get are long smearings without any band (I'm not sure whether this is called "smearing")
I've tried different primers and annealing temperatures
All of my primers have lower degeneracy than 200
I've still got the long smearings with no band
When I took the PCR products to reamplify again, there's no band nor smearing occurred
When I tried decreasing concentration of cDNA template, I've got faint smearing and almost couldn't see anything in the lowest concentration of template
I've read and found there're many possible factors affecting this
Would anyone suggest which should be considered first?
And could you please tell me how exactly to calculate Tm of degenerate primers?
Thank you very much for every suggestion
it looks to me that your primers were forming dimmers. I think you should redesign primers.