What can I do if the PCR generates errors?
What can I do if the PCR generates errors?
To avoid errors during PCR, we recommend using a high-fidelity enzyme (see selection guide). In addition, be sure to avoid the following:
1. Overcycling
Overcycling PCR reactions often:
*Changes the pH of the reaction in a manner that destabilizes DNA.
*Increases the amount of PCR product, thereby reducing the efficiency of the polymerase and promoting errors.
*Decreases the amount of dNTPs, thereby increasing the likelihood of base misincorporation due to the unbalanced dNTP concentration. (If using Takara Bio's PCR enzymes, the dNTP concentration is optimized to 200 nM; increasing dNTP concentration leads to misincorporation.)
*Causes accumulation of single-stranded and double-stranded DNA.
2. High Mg2+ concentrationMg2+ concentration ranges from 1–5 mM. Using a high Mg2+ concentration may increase yield, but it might also impact the proofreading activity of enzymes. However, the Mg2+ concentration should always be higher than the dNTP concentration.
3. Template DNA damageLimit UV exposure time when analyzing or excising PCR products from gels.