I wonder if anyone knows how active the EcoRI-HF (from NEB) is in the CutSmart buffer at 21 degC. The heating failed without me noticing and the reaction got incubated at room temp. (around 21C). I was digesting a PCR-generated fragment to be used in a Gibson assembly reaction for cloning purposes. I cannot tell the degree of completion from a gel, the size difference is too small. Is at least some portion of the fragments going to be digested? Thanks!
Just add another 0.5ul enzyme to your reaction and run at 37C overnight. This just in case it did not fully digest at 21C.
Thanks, Hanna. I unfortunately noticed the temperature issue too late into the process, not before I started the transformation. I can always digest with additional enzyme, as you said. I was more curious than anything, as I couldn't find anything on the activity at 21C.
I think it is better to be safe and just redo the digestion as your yields will probably be lower specially if you have to do a gel purification
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