Ssp 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.

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