I have digested my insert (gel purified PCR amplicon) with 2 restriction enzymes and kept for overnight incubation. After which I have put it in 4 degree without doing anything (phenol-chloroform extraction or heat inactivation of enzymes). a. Will there be over digestion? b. Will digestion still continue at 4 degree? c. Are restriction enzymes expected to be active at lower temperatures?
there will be activity at 4c but very greatly reduced. If your template has completely cut then there will be no further activity because all of the cut sites have been used and if you are using large volumes of cutting mix ( 20ul or more) you will not expect star activity which is often associated with just a few enzymes and excessive amounts of glycerol
Restriction digestion enzymes are known to be very efficient. And I think they can be active at 4 degrees. We in our lab stop the restriction digestion reaction by heating at 65 degrees for 10 minutes.
How many units of enzyme did you put in? Most enzymes are active at 37 and some at 25, they are not active at 4. Besides, if you did an overnight digestion by the time 24 plus hours have expired there should be little to no activity left, unless you added an excessive amount of enzyme. At that point there should be complete digestion and if there is star activity it's not because of the continued digestion, it's because you added too much. It should be fine to put your digest at 4 after an overnight but if you are worried you can put it at -20 too, that should stop all activity but not do anything to you digest. You can also clean up the digest by using a pcr purification spin column if you are just digesting a vector rather than running it out on a gel or phenol chloroformq
there will be activity at 4c but very greatly reduced. If your template has completely cut then there will be no further activity because all of the cut sites have been used and if you are using large volumes of cutting mix ( 20ul or more) you will not expect star activity which is often associated with just a few enzymes and excessive amounts of glycerol
In short I would put at -20c where things will be completely stable until continuing
- Badges
- Science method