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Pwo DNA Polymerase Troubleshooting

Product No. PWOPOL-RO

Troubleshooting

For general troubleshooting hints regarding PCR, please visit our homepage under http://www.roche-applied-science.com

There are a lot of parameters which could influence PCR fidelity negatively:

  • Organic solvents (like DMSO, ethanol, phenol; the latter two may be residues from DNA isolation),
  • Too long denaturating phase,
  • Too many cycles,
  • Too high concentration of dNTPs,
  • Impure or damaged template DNA,
  • Too high pH,
  • Non-optimal Mg2+ concentration.

In case you experience a decreased fidelity in PCR, a first measure should be to check the quality of template DNA and to perform a titration of MgSO4 to find the optimum concentration for fidelity. Usage of DMSO or other organic solvents should be avoided. If addition of DMSO is necessary due to a GC-rich template perform a titration to find the optimum concentration. Perform the PCR reaction with a moderate number of cycles (< 20) by increasing at the same time the input template DNA amount to achieve sufficient sensitivity.

Materials

     
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