That sounds easy, but often you have to play around with the conditions (enzyme conc/DNA conc/incubation time) to get a reasonable amount of partially digested product, using up a lot of precious time and DNA. Next you can run out the dilutions on a gel, find those that have hit the sweet spot to produce a reasonable band corresponding to the partially digested product, isolate the band and get on with the experiment. I’s a good idea to do the reaction in the PCR machine where you can set the time and temperature accurately then go straight into the heat-kill step with no fuss. Then you heat-kill the the enzyme after exactly 10 minutes. To do this you need to set up a series of digests with a fixed amount of plasmid DNA and a serial dilution of the restriction enzyme, starting with about 3-5 units of enzyme per microgram of DNA and making about 5-10 dilutions from there. ![]() Even normal restriction digests can go wrong, but partial digests are more tricky procedures that can take a bit of time to get right.Ī partial restriction digest involves performing an incomplete digestion of the plasmid DNA so that, in our example where you have two restriction sites for the enzyme in question, you will end up with three digestion products: one cut at both sites, one cut at the site you want and one cut at the site you don’t want.īut restriction enzymes work fast so to get a reasonable amount of partially digested plasmid, you need to play with reducing the amount of enzyme you put into the reaction, and the time of digest. A partial restriction digest is a plasmid-cloner’s last resort. The only restriction enzyme that cuts in a suitable position on your plasmid vector also, as luck would have it, cuts in another position elsewhere in the vector so you need to do a partial restriction digest to prepare your vector.Īt this point I would sympathise with you. You are planning your next cloning experiment, but there’s a problem. No matter how many times you look at it, it’s not going to change.
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