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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.

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Nucleotide concentration is too high or unbalanced The standard concentration is 20-200 M of each nucleotide Suggestions:
  1. Check the concentration of stock solutions of all nucleotides
  2. Double check the final concentrations of all
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Tan solid. A glycoside used to permeabilize cellular membranes. Exhibits hemolytic activity. Contaminants: ash: ≤10%. Soluble in H 2 O. pH 4.4 - 4.6. RTECS VQ1400000, CAS 8047-15-2. References:...
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... GelSpin Kit utilizes a silica based spin filter ... electrophoresis, the desired DNA band is cut from ... spin filter unit. The gel slice is melted ... the DNA is bound to the silica spin ...
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