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I have digested my DNA with NotI enzyme and put it in the −20 °C freezer without heat inactivating it. Can restriction enzymes work at −20 °C? Should I expect STAR activity?

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    $\begingroup$ I'd still recommend inactivating the enzyme or cleaning up the reaction after thawing up. $\endgroup$ – Nandor Poka Mar 19 '15 at 23:05
  • $\begingroup$ what is star activity? pls avoid acronms amap thnk y othrws t bcms unintellgbl $\endgroup$ – AliceD Mar 20 '15 at 2:41
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    $\begingroup$ @AliceD Star activity is a pretty common term. It refers to nonspecific digestion by restriction enzymes. $\endgroup$ – WYSIWYG Mar 20 '15 at 6:11
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    $\begingroup$ @AliceD Star activity is not an acronym, it is merely restriction enzyme jargon. -edit- ninjad. $\endgroup$ – March Ho Mar 20 '15 at 6:11
  • $\begingroup$ the capitals made it look like an acronym :) Thanks guys $\endgroup$ – AliceD Mar 20 '15 at 9:45
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Enzymes have temperature optima based on the organism they were isolated from. So I would predict the there is virtually no activity at −20 °C. Another consideration is that the reactions are likely to be frozen solid, so that would limit diffusion, and also slow it down. But the real question is: what are you afraid of? Just the star activity? Even if there were star activity, would that adversely affect your experiments?

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  • $\begingroup$ I do not know, I have never had star activity before. Your answer makes sense and I think like that too but I posted the question here because there was no answer to this on the internet and I thought maybe someone here has an experience on this. $\endgroup$ – ecagl Mar 19 '15 at 23:10
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    $\begingroup$ How about this paper? ncbi.nlm.nih.gov/pmc/articles/PMC2396408/pdf/gkn182.pdf $\endgroup$ – mdperry Mar 19 '15 at 23:18
  • $\begingroup$ @mdperry You should update that into your answer and perhaps include some excerpts from it, it is a great addition. $\endgroup$ – March Ho Mar 20 '15 at 6:14
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While there is unlikely to be any activity when the enzyme is frozen, there are better methods to prevent star activity.

1: If you are running the reaction in a PCR machine, you can program the machine prior to the digest to heat up to 65 degrees for 10 minutes after the intended digestion timeframe to heat inactivate NotI. Since NotI is a heat-labile enzyme, this prevents star activity by denaturing it.

2: You can use NotI-HF, which has been engineered by NEB to greatly reduce the amount of star activity and can therefore be left incubating at 37°C overnight.

3: You can gel purify or perform a "PCR purification" on your digested DNA samples, this also removes any protein present and therefore prevents star activity.

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  • $\begingroup$ @WYSIWYG Why the scare quotes, though? $\endgroup$ – March Ho Mar 20 '15 at 6:31
  • $\begingroup$ Additionally you can run the reaction in PCR tubes in the thermocycler. Simply choose the temperature you want to have it and then ramp up to 10 min at 65°C afterwards. No extra work needed and very convenient. $\endgroup$ – Chris Mar 20 '15 at 6:31
  • $\begingroup$ @Chris That was exactly what I meant. Sorry for being unclear. Edited answer. $\endgroup$ – March Ho Mar 20 '15 at 6:32
  • $\begingroup$ @Chris WYSIWYG edited the answer to "PCR purification" (you can check the log). I was unsure why this was formatted as such. $\endgroup$ – March Ho Mar 20 '15 at 6:33
  • $\begingroup$ Ah, now. I usually run these reactions in PCR tubes in the thermocycler, because the volume is small anyway and it is very convenient. $\endgroup$ – Chris Mar 20 '15 at 6:34

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