Fast digestion protocol

1 post / 0 new
swannnyy's picture
Fast digestion protocol

It's Friday afternoon, you have a hot date lined up, then your supervisor appears with a sample that they want digested and ligated into a vector. Today. Before you can go home.

Here's a quick protocol picked up at a conference a few years ago, in case you need to do a DNA digestion, but are short of time.
Rather than placing the tube in a waterbath, you can fully digest genomic DNA in 10-15 minutes, using a microwave. Here's how.

Prepare your digestion mix as usual. Place the reaction mix in a microwave, and heat for 30 seconds at medium to medium-low power (this applies for an older model microwave of about 600 to 800 W). Remove the tube from the microwave and place in water for 30 seconds.

The cycle is repeated for 10 to 15 minutes, and should yield well-digested DNA. Essentially all you are doing is raising the temperature enough to increase the reaction rate without denaturing the enzyme. Interestingly, the same idea also applies to ligation reactions. You can ligate cohesive termini in about 15 minutes.

(As an alternate strategy, you could try the same thing in a thermal cycler, going from 45 or 50 C down to 20 C. Please note, I have not attempted this particular expt, so the temperatures will probably need a bit of adjustement...)