Trouble-shooting: Misincorporation Or Low Fidelity

Here are some troubleshooting hints that we have gathered regarding misincorporation or low fidelity of PCR reactions:


		Magnesium concentration too high
		Nucleotide concentration is too high or unbalanced
		The pH of the reaction buffer is too high
		Mispriming caused by secondary structure of template, snapback, or excessive homology at 3' ends of primers
		Damaged template DNA

Non-optimal Mg++ concentration

Suggestion:Titrate magnesium concentration using our PCR Optimization Kit.

Nucleotide concentration is too high or unbalanced

The standard concentration is 20-200 µM of each nucleotide Suggestions:

The pH of the reaction buffer is too high

A pH 8.3 is optimal Suggestions:

pH of reaction buffer can be lowered to 5-6
Base substitution changes decreased by 60 fold and frame-shift changes decreased by 11 fold with a 3 unit pH decrease

Mispriming caused by secondary structure of template, snapback, or excessive homology at 3' ends of primers

Suggestions:

Increase initial denaturation temperature to 95-97°C
Denature DNA minus enzyme & buffer for 4-6 minutes
Increase cycling denaturation time 15-30 seconds
Try "Hot Start" technique
Add T4 Gene 32 protein 3-5 µl/ml
When designing primers, make sure there is no more than 2 bases of homology at the 3' end. Use a primer design program if available.
Consider addition of cosolvent to reaction buffer:
3-15% DMSO
1-10% Formamide
5-15% Polyethylene glycol
10-15% glycerol

Damaged template DNA

Suggestions:

Minimize cycle number, since repeated heating/cooling at high pH can damage template (Most templates require 25-35 cycles)
Check integrity of template DNA
Reduce cycling denature time to 15 - 30 seconds