Dear all, Im new here and I need help from you all.
How to inactivated restriction enzyme if the enzyme can't be heated inactivated? Such as MspI from NEB.
Please answer me. Thank you.
From NEB's web site:
"For enzymes that cannot be heat-inactivated, we recommend using a column for cleanup (such as the Monarch® PCR & DNA Cleanup Kit), or running the reaction on an agarose gel and then extracting the DNA (we recommend Monarch Gel Extraction Kit), or performing a phenol/chloroform extraction."
I notice that all the restriction enzyme buffers include 10 mM Mg2+, which suggests that these enzymes are Mg2+-dependent. In that case, it should be possible to inhibit them by chelating the Mg2+ by adding a molar excess of EDTA, say 20 mM.
Restriction MspI NEB inactivation
What is your objective to inactivate MspI? I believe that you want to continue DNA restriction with another enzyme or PCR reaction…
You can do phenol/Chlorform protein precipitation and continue with Ethanol DNA method, centrifuge and recuperate the DNA in the pellet and so make DNA solution in TE Buffer.
If you can use DNA extraction from your MspI mix with Qiagen kit protocol that may eliminate proteins and restriction enzyme so you recuperate your DNA and go on other reactions: Restriction, PCR or Cloning
However these protocols could better success with a significant and substantial amount of DNA: 5 to 10 micrograms.
65C for 15min will heat inactivate your respective enzyme, as well as you can add Etbr directly post incubation .
Good luck
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