Microwave restriction digests
Prenom.Nom at rsvs.ulaval.ca
Thu Sep 22 05:07:08 EST 1994
In article <jpcd0-2009942104500001 at macr1-1.welc.cam.ac.uk>,
jpcd0 at mole.bio.cam.ac.uk (John Dixon) wrote:
> Someone in our lab heard about this and put set up two digests: both had
> the same amount of enzyme; he put one in the m/wave at full power for a
> minute as I remember and 1 at 37 deg C for a minute. Much to our surprise
> both had virtually cut to completion. The unmicrowaved one had gone
> slightly further.
> We decided that the enzyme used was probably more efficient than the
> manufacturer stated and that 1ul of approx 10u/ul enzyme (as listed and
> maybe conservatively estimated) perhaps would cut 100ng or so clean DNA in
> a minute or two (considering that the defn of an enzyme unit is that it
> cuts a ug DNA in 1hr).
> Did you run an unmicrowaved control?
No, I just tried out the procedure to see how well it works. I have
heard that without incubation at 37 degrees or microwaving, there is little
cutting after several minutes. So far, I've only used plasmid DNA which
has only a few cutting sites at most. But, a couple people in the lab have
digested total DNA with very successful results.
>I'm glad Blue posted this today, because I suddenly developed a good reason
>to try it! Because of 'operator error', I suddenly needed to get a
>restriction digest done in a hurry, so I tried this microwave digestion
>I digested 2.5micrograms of plasmid with 20 units of SacI and HindIII in a
>volume of 20microliters. Our lab has a 650W microwave with a rotating
>tray. I heated for 15 seconds maximum power, followed by 2 minutes at room
>temp. I repeated this four times as Blue suggested and then ran on a gel.
>The digestion worked very well.
Apparently, the conditions for the digestions can vary somewhat. From the
four x 15 sec (650 W), 2 min rest period OR
four x 10 sec (900 W), 2 min
one x 1 min (??? W)
Anyone else out there have any experiences with this method?? Blue
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