Frequently Asked Questions

Q- Will the type of PCR enzyme also affect specificity and yield in PCR ? If so, then which enzyme should I use to optimize my reaction?

A- PCR enzyme selection can be critical to PCR performance. Taq DNA polymerase has a higher error rate (no proofreading 3’ to 5’ exonuclease activity) than Pfx or Pfu. Use Pfx if high fidelity is needed. However, if processivity and/or the addition of an adenine residue to the 3’ end of the PCR product is desired for downstream cloning, then Taq may be preferable.


Q- You mentioned the importance of Tm for a successful PCR experiment. What about number of cycles used for the reaction?

A- For a typical PCR with three temperature steps, thermal cycling rounds are usually set to a number between 25 to 35. Increasing the number of cycles above 35 will result in a greater quantity of PCR product, but it will often result in the enrichment of undesirable secondary products. On the other hand, too few cycles will give low product yield.

 

Q- I often see a small product of less than 100 bp on the agarose gel; how do I get rid of this?

A- The small product is likely to be a primer-dimer that has formed during PCR. To minimize primer-dimer formation, alter the ratio of template to primer. If the primer concentration is in extreme excess over the template concentration, then the primers will be more likely to anneal to themselves or each other instead of the DNA template.

 

Q- How does the purity of template DNA affect PCR optimization?

A- DNA quality and purity has a significant effect on the success of a PCR experiment. Common DNA extraction reagents that inhibit PCR include protein, RNA, organic solvents, and detergents. The ideal ratio of A260/A280 is between 1.8 and 2.0. A lower reading indicates protein and/or solvent contamination.

 

Q- I need a thermal cycler, but what cycler specifications should I look for?

A- There are three critical specifications that you should consider when looking for a new thermal cycler. The first is temperature accuracy, which is a measure of how close the actual temperature in a given well is to the programmed temperature. Even small deviations between the programmed and the actual temperatures can make a difference in yield and specificity. The second is temperature uniformity, which is a term used to indicate how much temperature variation there is across the thermal cycling block. Lower-specification cyclers will exhibit more temperature variation at higher temperatures, which can lead to inconsistent results. The third is speed (or ramp rate), which is the time the thermal cycler takes to transition from one programmed temperature to the next. Differences in ramp rate can influence the course of the reaction. Sample ramp rate is a more accurate reading than ramp rate, because it measures the temperature of an individual reaction, not the block.

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