Dear All,
I did the extension experiment with 20mer RNA primer annealing with 40mer RNA template in the presence of recombinant human coronavirus SARS-CoV-2 RNA-dependent RNA polymerase (RDRP) which was purchased from the vendor. No extension product was observed but some products which were lower than primer shown were found (please see the attached screenshot). Does anyone know the possible reason to cause this? The primer and template were purchased directly from IDT and they are still good (I checked the purity via HPLC). And the polymerase was purchased in July and are stored under -80C until using. Thanks in advance!
RNA Polymerase should have 3’-5’ exonuclease proof reading activity and it is simultaneously degrading the primer and extending it with ATP. If the exonuclease site probably glutamates and aspartates that bind divalent magnesium are mutated to alanine this should knock that out.
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