I'm looking to perform a restriction digest using KpnI and BglII however all the suppliers I look at (Promega, NEB, etc) all have the two enzymes operating in different buffers. Does anyone know if these two enzymes can be used in the same buffer? If so are there any companies that will supply these two enzymes in the same buffer? Or would I be better off doing one digestion followed by the other?
Hello Shaun,
you could use BglII together with KpnI by choosing the FastDigest enzymes from Thermo Scientific:
http://www.thermoscientificbio.com/restriction-enzymes/fastdigest-bglii/
http://www.thermoscientificbio.com/restriction-enzymes/fastdigest-kpni/
For both the same buffer and the same temperature (37°C) can be used. But you should keep in mind that BglII can't be heat-inactivated at 80°C (which is possible for KpnI).
I hope this information helps you.
Best regards!
In my experience, double digests are usually possible; check the manual from your suplier on which buffer to use. For this particular combination, KpnI is probably the most difficult to handle, so preferentially optimize conditions for this enzyme. It also seems to go bad very quickly, even when stored correctly.
It is always helpful to make single digests in parallel, just to check whether your enzymes are active under those conditions.
I've done double digests in the past, so I know they can be done effectively, I just needed to know if it was possible with these enzymes. Thanks all for your help, all the best, Shaun.
New England Biolabs has a pretty convenient double digest finder showing which of their enzymes can be used together (https://www.neb.com/tools-and-resources/interactive-tools/double-digest-finder). They've modified a lot of them to be compatible with their CutSmart buffer
I actually did something similar last week, if you're using the NEB buffers start with the lower number (i.e. the lower salt concentration). For example, I was doing SacI and BglII, so I did a 20uL digest with SacI first (NEB Buffer 1), then let it incubate for an hour, then add 4ul H2O, 3uL of NEB Buffer 3, 2uL of BglII, and 1ul of 1ug/ul BSA to make it a 30ul reaction and then let incubate for another hour.
We do this all the time in our lab and it works great
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