Double digest question for ligation - (Apr/03/2007 )
This may be a very simple question. I need to do a NotI/EcoRI double digest on my insert and vector. The ligations haven't been working. I recently was told by a lab tech that a NotI digestion of a PCR product may need to go overnight. Can I throw in the NotI/EcoRI double digest mix in together and just incubate it over night? Or should I digest with EcoRI first, clean up the reaction, then overnight digest with NotI? If I'm having NotI digest problems on my PCR product, would I be having NotI digest problems for my vector too?
Yes, you can throw them together, as far as I remember Eco RI and Not I shouldn't make problems in a double digest. But be sure to keep the content of glycerol lower than 5% in your reaction mix.
You also can cut with Eco RI over night and then add Not I the next day without purification (don't forget to add buffer and water also to get the right concentrations again).
It should work fine (see http://www.neb.com/nebecomm/DoubleDigestCalculator.asp). Just wondering how far from the end of your PCR product you have placed your restriction sites?
12 bp from the end.
if both the sites have adequate bases on either sides, you should have complete digestion of the PCR in an hour.