Hello,
I am using TriLink's Mutagenesis dNTP mix to perform EPR on a 1.3 kb fragment. I have been unable to observe a PCR product when following their recommended cycling parameters. I am able to successfully amplify my product using Phusion so my template and primers seem to be fine.
Cycling Conditions:
95 C - 3 min - Initial denaturation
92C- 1 min/53 C - 1.5 min/72 C - 5 min x 30
72 C - 10 min - Final extension
Components: 10x PCR buffer, 500 nM Forward/Reverse Primer, 10 fmol template, Mutagenesis dNTP mix, 2 mM MgCl2, Taq (5U/uL), water up to 25 uL
Any troubleshooting tips?
Check out Mg concentration? Probably the kit concentration is to high. For long PCR amplifications I use 2 mM.
Hello Sushanth
Try a gradient PCR (48,50,52,54,56,58) and see if their is any amplification.
- Generally speaking phusion is a highly processive enzyme so will always amplify larger fragments - that is fragments > 1kb - with higher efficiency than nearly all other polymerases
- In terms of your alternative method with validated template and primers I would do the following
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