PCR primer Tm and concentration question - (Jun/13/2005 )
Hi all
greetings
iam working with the isolation of RNA,cDNA synthesis and PCR of a eukaryotic gene.But iam not getting the product .so can anybody tell me how to find the annealing temperatuere for the primer. what is the idael concentration of primer that can be used to get a good result.
Hi!
the best way to calculate annealing temperature is to calculate melting tempoerature using the formula:
Tm (in deg c)= 81.5+0.41(G+C%)+16.6 logNa - 500/length of primer
Move according to the concentrations mentioned on the oligi synthesis report. what ever the Tm comes, put up the gradient for annealing to
5 degrees . hopefully u will be able to get ur band.
All the best.
greetings
iam working with the isolation of RNA,cDNA synthesis and PCR of a eukaryotic gene.But iam not getting the product .so can anybody tell me how to find the annealing temperatuere for the primer. what is the idael concentration of primer that can be used to get a good result.
I guess your problem is not just the annealing temp but may also be the low abundance of your target gene.
hi*
agree to tweety. But for primers up to 20nt Tm may be also calculated but this fomula : Tm = 4 x number of G/C + 2 x number of A/T