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 [...]

How can I decontaminate if I have PCR contamination?

-Leave pipettes under UV light in the cell culture hood overnight. UV irradiation promotes cross-linking of thymidine residues, damaging residual DNA. -Spray workstations/equipment/pipettes with 10% bleach and then wipe clean. -Change workstations; move the pre-PCR area to another pre-cleaned location. -Do not use any instruments or pipettes you have used before.PCR [...]

What are some sources of PCR contamination?

There are four main sources of PCR contamination: -The most common source of contamination is PCR product from previous amplifications (called ""carryover contamination""). When large amounts of PCR product (1012 molecules) are generated repeatedly over a period of time, the potential for contamination increases. -Another source of contamination is cloned DNA previously handled in the [...]

If PCR generates a smear after running the products on a gel, what can be done to improve the results?

First, determine the source of the smear using positive and negative (no template) controls. This can determine if the cause of the smear is contamination or overcycling, or if the smear results from poorly designed primers or suboptimal PCR conditions. If the negative control is blank, there is no contamination. Instead, the PCR conditions will need to be optimized; consider the following when [...]

If there are nonspecific amplification bands, what can be done to improve specificity?

1. All Takara Bio PCR polymerases -Issue: Primers are not specific. -Solution: Use BLAST alignment to determine if the 3' ends of the primers are complementary to sites other than the target site(s). Redesign primers if necessary or modify PCR conditions. -Issue: PCR conditions are not sufficiently stringent. -Solutions: *Increase the annealing temperature in increments of 2 degrees. *Use [...]