I am working on a virus. trying to clone the 2 genes , one gene i amplifies by using specific primars, but with other gene i am not able to amplfy it . i have tried it at least 10 times, more than that i designed new primar also but didn,t get the band . i want to know were can be the problem.
i tried it with 30 ul reaction volume, 20 ul rxt volume, 50ul rxt volume.
also i used to add tag polymerase at the end
In case of reaction mixture , i use the same constitutents as used with my other gene & also the in same concentration .
Ialso checked it at different annealing temperatures. and at different concentrations of pcr reaction mixture constituents.