PCR Tips & Tricks

Most of you are probably aware of the common variations and tweaks you can do to optimize a PCR reaction, such as changing the MgCl₂ concentrations. But if you are needing to amplify something tricky, such as a transcript with high GC content for cloning, PCR optimization can become a nightmare.

Tip #1 – Re-amplification

When a nested PCR is out of the question, a re-amplification works just as well. The principle is similar to a nested PCR in that you are enriching the region of interest.

Simply run your PCR reaction in a thermocycler for 10-15 cycles and take 0.5ul of the completed reaction to use as template for a second set of reactions.

Tip #2 – Ramping

In some instances where it is difficult to get your primers to hybridize to the correct complementary sequence of your template (e.g. when amplifying a GC rich region, cloning cDNA that has GC or AT strings, etc), a great technique to try is to lower the ramping speed from the denaturation to annealing steps as well as the annealing to extension steps.

The ramping speed is the time it takes for the thermocycler to heat/cool the reaction to a different temperature i.e. to go from 95 degrees to 60 degrees. For instance, by lowering the ramping speed from 100% to 20% you are essentially giving your primers time to hybridize to their complementary sequences on the template. The principle underlying this technique was inspired by the oligo-annealing step of an EMSA protocol.