hi, i am doing nested pcr these days but my outer primers are not giving bright required band but other non-specific dense bands. I am using master mix recipie for 25 microliter pcr reaction as":
Nuclease free water 13.6 ul
Buffer 2.5 ul
25 mM MgCl2 2 ul
2mM dDNTPs mix 2.5 ul
Primers 2ul
DNA 2ul
Taq poly 0.4 ul.
Hii.. I think so you should 1) check out the annealing temperature which might not be sufficient for the primer binding to DNA...or 2) decrease the concentration of dNTP to prevent the non specific binding...3)be sure that the DNA is RNA free or not contaminated with any other agent otherwise the concentration of DNA would be too low for PCR..!!!
Hope that help you..all the best!!
Hii.. I think so you should 1) check out the annealing temperature which might not be sufficient for the primer binding to DNA...or 2) decrease the concentration of dNTP to prevent the non specific binding...3)be sure that the DNA is RNA free or not contaminated with any other agent otherwise the concentration of DNA would be too low for PCR..!!!
Hope that help you..all the best!!
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