If no amplification products are obtained, what parameters should be considered first when troubleshooting?

1. Consider the following: -First, ensure that all PCR components were included in the reactions. A positive control should always be included to ensure that each component is present and functional. -If there were no problems with the experimental setup, increase the number of PCR cycles (3–5 cycles at a time), up to 40 cycles. Increasing the cycle number can overcome issues with a [...]

What is the role of salt in PCR reactions?

Successful PCR requires that the DNA duplex separates during the denaturation step and that primers anneal to the denatured DNA. Salt neutralizes the negative charges on the phosphate backbone of DNA, stabilizing double-stranded DNA by offsetting negative charges that would otherwise repel one another. Potassium chloride (KCl) is normally used in PCR amplifications at a final concentration of 50 [...]

What is the role of magnesium in PCR, and what is the optimal concentration?

Magnesium is a required cofactor for thermostable DNA polymerases and is important for successful amplification. Without adequate free Mg2+, PCR polymerases are not active. In contrast, excess free Mg2+ reduces enzyme fidelity and may increase nonspecific amplification. A number of factors can affect the amount of free Mg2+ in a reaction, including DNA template concentration, chelating agents in [...]

How can I optimize PCR conditions for AT-rich templates?

Some templates may have long AT-rich stretches that are hard to amplify under standard reaction conditions. The Plasmodium falciparum genome is about 80% AT, and regions flanking genes are often AT rich. Polymerases recommended for GC-rich templates, such as EmeraldAmp GT PCR Master Mix, EmeraldAmp Max PCR master mixes, and PrimeSTAR GXL DNA Polymerase, are also suitable for AT-rich templates. [...]

Can DMSO be added to improve amplification of GC-rich templates?

We have heard from customers that improved amplification of GC-rich templates was obtained by adding DMSO to reactions using PrimeSTAR MAX DNA Polymerase or CloneAmp HiFi PCR Premix. The recommended concentration of DMSO is between 2.5% and 5%.PCR [...]