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Note about PCR protocols

Posted by on Wednesday, May 10, 2017 in News.

The PCR lab lists two different protocols depending on your preferred Taq polymerase. Each protocol has an optimal annealing temperature for PCR.

Main Protocol

The main protocol uses Taq polymerase from either Fisher (K1081) or NEB (M0484S). With these reagents, the best annealing temperature is 49°C. If you use a higher annealing temperature (55°C), only the Wolbachia band will be visible. The CO1 band may not be visible.

If you are setting up a reaction with only Wolbachia primers and not the CO1 primers, you may use the old annealing temperature (55°C) and it will give a slightly brighter band for Wolbachia.

Alternate Protocol

The alternate protocol uses GE Illustra PureTaq PCR beads. A 55°C annealing temperature works best with this reagent.

Other Taq Polymerases

You may run this lab with a different Taq polymerase. If you use a different reagent, please test it beforehand to make sure you get the expected results. If you don’t get expected results, try using a different annealing temperature, between 45°C and 55°C. The denaturation and extension temperatures should remain the same.