Ahh, PCR, Kary Mullis’s Nobel Prize-worthy, LSD-inspired copy machine for DNA. He dreamed it up while driving down the Pacific Coast Highway, and now it’s used for everything from detecting SARS-CoV-2 to crime scene investigations to sequencing dinosaur DNA. Here are some tips I’ve picked up over the years:
- Set up a 50 µL reaction, then split it into two 25 µL reactions. Run one at Tm, the other at Tm – 5 ºC.
- Fool around with different DMSO concentrations. I have lately taken to using sulfolane (1-3%), an alternative to DMSO that may work better. Occasionally, sulfolane-DMSO mixtures have helped.
- Play around with primer designs. Use a G or C at the 3′ end when possible to anchor it down (the “GC clamp”). Use an online tool to check for pesky self-complementarity, and tweak the primers to keep the Tm values close together.
- Even if you don’t have fancy hot-start reagents, you can still manually hot-start a few PCR reactions at a time by adding the polymerase once the samples are up to temperature for the initial 98 ºC denaturation step. This is easier if you are adding the polymerase as a 5 or 10 µL solution in 1x buffer. (Hot start prevents annealing to off-target sequences in the important initial round of amplification. It also can improve yields and prevent primer dimerization).
- With the commonly-used Phusion high-fidelity polymerase, GC buffer works far better than HF buffer in my experience, despite the manufacturer’s recommendations to use HF first and GC only for difficult templates. Note that the error rate is twice as high for GC (9.5 x 10-7 versus 4.4 x 10-7 — a big relative change of 2.2-fold, but still small when the absolute rate is considered). The yield is much higher with GC buffer, and I sometimes don’t even get bands using HF.
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Med School Beast 