January 1, 1970 (Vol. , No. )
Tom Russell, Ph.D. Sigma Life Science
Eric Reyes Sigma Life Science
Successful PCR runs require more than excellent primer design. Here are seven best practices for both obtaining consistently high-quality data and ensuring the integrity and economic use of your PCR reagents.
- Centrifuge lyophilized primers upon delivery. While the DNA is usually present as a nearly invisible film on the bottom of the tube, it can come loose and fall out when the cap is removed for the first time.
- Resuspend primers in 10 mM Tris pH 7.5, 1 mM EDTA solution (TE buffer) instead of water. The Tris and EDTA prevent acidic water and contaminating DNases from hydrolyzing and enzymatically degrading DNA, respectively.
- Aliquot the resuspended primers into working stocks. This eliminates the need for damaging freeze / thaw cycles of the master stock as the working stocks can be removed from the freezer three to five times without degrading the DNA. If contamination of a working stock occurs, it can be thrown out and replaced with another without compromising the master stock.
- As with your primers, if you purchase large volumes (e.g., 50 mL) of 2X PCR premixes, you can aliquot these reagents into smaller working stocks that are suitable for a single experiment (e.g., a 96-well plate) to avoid contamination.
- For each target gene you are working on, make a “master mix” of your 2X PCR premix, water, and primers. Some people factor in extra replicates or a percentage (~10%) of volume for each master mix to account for pipetting error. You can then add this “master mix” to the tubes/plates first and then add your samples.
- If you want to be “extra-passionate” about the precision between your technical replicates, you can even make a “master mix” for each target and sample—add the 2X PCR premix, primers, water, and template—and then add everything all at once into your tubes/plates. This will require extra tubes, but the intra-replicate variability can be greatly reduced.
- Always keep your pipettes calibrated and take your time. Reagents are expensive, and samples can be limited.
Tom Russell, Ph.D., and Eric Reyes are global product managers for oligonucleotides and PCR Reagents, respectively, at Sigma Life Science. To learn more about Sigma Life Science’s PCR reagents and online oligo design tools, click here.