Hello everyone, I have had problems performing a double DNA digestion with restriction enzymes AscI and FseI. When I perform the digestion with each enzyme separately, they linearize the DNA to the expected molecular size (they work correctly) but when I double-digest with both enzymes together they only linearize the DNA, do not extract the expected band. Can anyone help me with this?
It sounds like this would be a good candidate for sequential digestion since both enzymes are working separately. If you can heat inactivate (yes, see below) and clean up one or both digestions to get rid of the enzyme and buffer--either by using a clean-up column or by phenol:chloroform extraction and ethanol precipitation (the old school method)--you can then elute your DNA, add the second buffer, and then the second enzyme. The second enzyme should work as well as it did in the initial, single digestion unless their is something else going on. Since you're expecting to see an additional band, it doesn't sound like you're trying to cut sites that are too close together to both work.
Another trick that is possible is if the two enzyme buffers are very similar except for salt concentration. In that case, you can digest with the enzyme that uses low or no salt buffer until completion, heat inactivate the 1st enzyme, add concentrated salt together with more low-salt buffer (in order to keep the buffer at 1X), and then the second enzyme and digest to completion. Both enzymes (from NEB) are said to be capable of heat inactivation, so that is helpful. AscI looks to be strongly inhibited by certain salts, so it would probably have to be used first if FseI needs salt in the buffer. Good luck.
According to the NEB website, you should do a double-digest in their CutSmart buffer
https://nebcloner.neb.com/#!/protocol/re/double/FseI,AscI
It also says that these enzymes are sensitive to methylation status. See the link for more info.
If they each work, I would not suspect problem with methylation.
Can you provide more details on the size of expected fragments, etc. and to show some results?
Thank you very much for answering. Your comments have been very useful. I'm going to put some of your advice into practice.
- Badges
- Science topic
- Similar topics
- Phytochemistry
- Extracts