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We have a long protocol that we are optimizing that includes DpnI digestion of a PCR product (to remove any of the template DNA if it's methylated, and while we're not certain in the blind tests, likely relevant DNA will be methylated).

I'm wondering if we can extend the DpnI incubation/reaction time to 4-5 hours. This will make concurrent steps in the screen easier to manage. Has anyone extended DpnI digestions? How "leaky" or nonspecific of a process can it be?

Thanks.

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  • $\begingroup$ We used DpnI a lot for eliminating "old" plasmids after PCR based site directed mutagenesis. The incubation time varied between 1-5 hours (depending on what else needed to be done) and I never had problems with getting clones from these reactions - and never had trouble with false positives due to incomplete digestion. $\endgroup$
    – Chris
    Commented Aug 12, 2014 at 21:31
  • $\begingroup$ If you take your DpnI digest tubes out of the 37oC water-bath after the instructed time (for me its after 5 minutes for PCR based site-directed mutagenesis) and either put at RT or preferably on ice, then you should be fine since the enzyme will not be functioning at those temperatures! $\endgroup$ Commented Aug 12, 2014 at 21:32
  • $\begingroup$ @Bez I'm trying to avoid having the person travel back to the room where the PCR machine is (from an animal facility), thus my question is more in line with Chris's response (I want to know if leaving it longer will be an issue). $\endgroup$
    – Atl LED
    Commented Aug 12, 2014 at 21:47
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    $\begingroup$ According to this instruction (chem.agilent.com/library/usermanuals/Public/200521.pdf) it can be done for 1 hour but this is for large plasmids. I agree with Chris that it should not be of a problem. Alternatively you can setup your 37oC DpnI incubation step in a PCR machine and after however long that is recommended, you can set the PCR machine to cool to 10oC automatically, that way you can leave it there for as long as you want. $\endgroup$ Commented Aug 12, 2014 at 21:57
  • $\begingroup$ @bez good point. While I hate to keep PCR machines at low temps for extended periods of time (causes more stress on the machine), dropping it to 4C after an hour is probably the best bet. Make it answer and I'll accept. $\endgroup$
    – Atl LED
    Commented Aug 13, 2014 at 13:22

3 Answers 3

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According to this instruction it can be done for 1 hour but this is for large plasmids. I agree with Chris that it should not be of a problem. Alternatively you can setup your 37°C DpnI incubation step in a PCR machine and after however long that is recommended, you can set the PCR machine to cool to 4°C automatically, that way you can leave it there for as long as you want.

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My mentor taught me that we can do DpnI digestion for minimum 4 hours and maximum 12 hours. I have ever left the reaction until around 16 hours (?) but the result was okay. My mentor recommended to put at 4oC after 12 hours, if we cannot continue the work immediately.

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  • $\begingroup$ can you add any references to your answer? also, can you check your answer for typos - is "ever" supposed to be "never"? $\endgroup$ Commented Jan 9, 2017 at 12:53
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In the lab I'm training in, we generally add small quantities of enzyme and keep it for overnight digestion at 37 deg. So the amount of time you want to keep it for depends on the amount of enzyme you're adding. Check the units of the enzyme and add accordingly to the amount of your DNA.

We did a DpnI digestion for mutagenesis too, we added 100 ng of our plasmid with 1uL enzyme and it worked decently. We had another set with 2uL DpnI but that doesn't seem to have worked out since we had zero colonies.

Probably the excess enzyme caused a loss of specificity or some sort of negative feedback inhibition.

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  • $\begingroup$ Sharing lab practice is great, but preferably add your sources (links, books, papers,reference works (Maniatis?)) so that folks can background-read on your topic. $\endgroup$
    – AliceD
    Commented Mar 31, 2017 at 14:24

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