Absence of target gene of nested PCR

mushy

New member
Nov 16, 2015
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#1
I carried out a nest 2 PCR on the positive control through gradient PCR. However, there is no band observed after the gel electrophoresis, but there is primer dimer. Due to limited stock of DNA template, may I know what is the best way to troubleshoot the problem? The total PCR reaction mixture I run for Nest 2 PCR

is 20 microlitre with 2 microlitre of DNA template obtained from the Nest 1 PCR.


 

Ruderus

New member
Nov 16, 2015
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#2
If you really have primer dimers, you can try to add the primers after the initial denaturation... this will avoid that they form dimers while you set up the reaction.

You can also try to lower the primer concentration and increase the annealing temperature.



Also, have you checked on gel the nest 1 ? do you have dimers there too? if not, maybe try to use more nest 1 template... if you have dimers there too.... well .... maybe redisgning everything will be easier than troubleshooting!



Good luck, I hope this helps!