Hinf I - FAQs

I need to digest my plasmid with 2 or 3 different restriction enzymes. Can I perform a simultaneous double or even triple digestion?

If you are able to find a buffer in which all 3 enzymes have sufficient activity (usually not lower than 50%), you can set up a single digestion with all 3 enzymes. It is important that the total volume of enzymes you add to your reaction is not more than 1/10th of the total reaction volume. The reason for this is that some enzymes have star activity if the concentration of glycerol exceeds 5%. If you are not able to find a buffer in which all your enzymes have sufficient activity, you will have to perform sequential digestions of the plasmid with the individual enzymes.

How can I check the activity of a restriction enzyme?

Here are some recommendations:
1. You can verify the restriction endonuclease has activity by digesting the unit substrate (i.e., lambda or Ad-2 DNA) using the reaction conditions identified for unit definition: one unit of the restriction endonuclease should digest one µg of the unit substrate in one hour in the appropriate reaction buffer at the appropriate temperature.
2. You can test for the presence of inhibitors of restriction digestion in the sample DNA. Perform a restriction digest in which some of the sample DNA is included along with some of the unit substrate (i.e., lambda or Ad-2 DNA). If digestion of the unit substrate occurs alone but is not observed when the sample DNA is added, then a diffusible inhibitor of restriction digestion is present in the sample DNA. If digestion of the unit substrate occurs in the presence of the sample DNA, but the sample DNA is not digested, then the failure of the restriction endonuclease may be due to sensitivity of the restriction endonuclease to methylation in the sample DNA.
3. Finally, to identify any tube-specific issues like shipment or storage stability problems, you can test function by performing a side-by-side reaction with a different lot of the same Invitrogen restriction endonuclease and comparing results.

Which restriction enzymes can exhibit star activity, and what factors contribute to star activity?

Star activity can be seen with many enzymes. See below for a list of some enzymes for which star activity has been reported. Some suggested causes for star activity include high glycerol concentrations, incorrect NaCl concentrations, and Mn2+ being substituted for Mg2+.

Some known enzymes that can exhibit star activity:
AdeI, AfaI, AloI, Alw26I, ApoI, AseI, AvaI *, AvaII, BamHI *, BanI, BanII, BceAI, BclI, BfmI, BfuI, BglI, Bme216I, BplI, Bpu10I, BseXI, Bsh1285I, Bsh1365I, BshTI, Bsp68I, Bsp143I, Bsp143II, BspMI, BsrBRI, BssHII, BstI, Bst1107I, BstEII, BstPI, BstXI, BsuRI, CeqI, CfrI, Cfr9I, Cfr10I, CviJI 2843, Eam1105I, Eco31I, Eco32I, Eco57I, Eco72I, Eco105I, EcoKMcrBC, EcoO65I, EcoRI *, M.EcoRI, EcoRV *, EcoT22I, EheI, FbaI, HaeIII *, HhaI *, HindIII *, HinfI *, HpaI *, HphI, KspAI, MamI, MbiI, MboII, MluI *, Mph1103I, MspA1I, MunI, MvaI, NciI, NcoI *, NcuI, NdeI *, NgoMIV, NheI *, PagI, PauI, PdmI, PmeI, PpiI, PshBI, PstI *, PsuI, PvuII *, RsrI, SacI, SalI*, Sau3AI, ScaI *, SchI, SdaI, SduI, SfiI, SgfI, SgrAI, SmaI *, SpeI *, Sse838, SspI *, SstI *, SwaI, TaqI *, TatI, Tth111I, TthHB8I, Van91I, XapI, XbaI *
* sold by Thermo Fisher Scientific

I would like to perform a restriction enzyme double digestion. Do you offer a tool to determine optimal reaction conditions?

Please use our DoubleDigest Calculator linked below: https://www.thermofisher.com/us/en/home/brands/thermo-scientific/molecular-biology/thermo-scientific-restriction-modifying-enzymes/restriction-enzymes-thermo-scientific/double-digest-calculator-thermo-scientific.html