I have done a digestion with SmaI (NEB) and did not realize the optimal temperature for the reaction is 25 degrees. I left it at 37 degrees for more than 1h, and I got the following pattern:
~600bp
~3800bp
~6500bp
and what I was expecting was:
~4200bp
~6500bp
As the pattern suggest, the expected band of 4200 seems to be being cut into 600 and 3800. That`s why I asked this question.
thank you
Hi there,
37°C is only detrimental to the SmaI enzyme stability. I've never heard about temperature triggering star activity. Have ou run uncut and cut samples in parallel?
Hi,
I agree, 37˚C should just decrease enzyme stability. In fact, NEB says that the half-life of the enzyme at 37 is only 15 min, so star activity seems unlikely...
Any chance you got a mutation that introduced a cut site at this "off-target" position? Do you have anything similar to CCCGGG anywhere else in your sequence?
thank you for your answer Dominique and Marcelo.
I have not run uncut and cut samples in parallel, could do. But i could also be a mutation, since I have done PCRs.
thank you very much again
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