Primers for Real time PCR - Really need some advice (Feb/20/2008 )
Recently, I make 9 pairs of primers to do my SYBR green Real time PCR.
These two pairs of primers below can amplify the DNA, and the product size are in the right position using 50bp DNA marker.
But the melting curve seem not very good, there are other bands on the lanes. The unspecific bands are about 50bp.
The annealing temperatures are both 56=C.
I really need some advice to improve my primers or the procedure of Real-Time PCR.
Forward1：ACC AGA CGC CTT CAA TCC TG
Reverse1：aga ggc ggg gac act gaa tga c
F2：TCT CCA TGA CCC ACA CTA CTT TG
R2：TGG AGG ATG GTG GTG AAG AAG
my first advise is to increase the annealing temp to 60°C or do a gradient PCR. furthermore, you can also try to decrease primer concentrations and annealing/extension time.