I would like to receive some feedback from people who have some experience with restriction digestion and DNA purification.
I set up a digestion of a plasmid that is 8 kb in size. I digested 3 ug of the plasmid in a 40 ul Rx, using 12 units of AvrII restriction enzyme (cuts my plasmid only once). I ran the Rx O/N. Then, I proceeded to run 2 ul of the digest in a 1% gel in order to see that my DNA was cut (uncut DNA will run much lower than my 8 kb marker; I also ran an uncut control in a different occasion which confirmed that the uncut plasmid ran faster in a 1% gel). My gel picture showed me a clear sharp band at 8 kb with no smears or anything else that would indicate star activity or DNA degradation, so I was confident that my DNA was properly cut.
After this, I proceeded to purify my cut DNA using a PCR purification kit without heat inactivation of the enzyme. Previously, this same procedure has yielded in 50 - 60 % DNA recovery in the past when I used StuI restriction enzyme (also a single cutter for this plasmid) instead of AvrII. However, when I applied the same procedure to the AvrII cut DNA, my recovery was of 10%, which gave me insufficient DNA for subsequent experiments.
I would like to know if anyone in this forum has experienced something like this. I re-measured the DNA concentration from the purified DNA, as well as the original uncut plasmid stock, and it always gave me the same DNA concentrations. More specifically, I would like to know if anyone here knows whether heat inactivating a restriction enzyme will increase the DNA recovery.
Thank you all for your time.