Microwave restriction digests

John Dixon jpcd0 at mole.bio.cam.ac.uk
Tue Sep 20 15:02:29 EST 1994


In article <Prenom.Nom-190994191534 at agrobacterium.rsvs.ulaval.ca>,
Prenom.Nom at rsvs.ulaval.ca (Prenom Nom) wrote:

> An interesting way of speeding up your restriction digests.  Make up your
> digests as usual in a volume of between 10-50 uL.  Then microwave at high
> power for four times for 10 sec with 2 min intervals at room temperature. 
> Then, run on a standard agarose gel.  I was very surprised that it worked. 
> Apparently, the power of the microwave makes a difference.  We have a 900 W
> oven.
>      What I don't understand is why it works at all.  Most enzymes are
> deactivated at elevated temperatures.  Ten mLs of water is very hot after
> 10 secs in the oven, and imagine a 50 uL reaction using the same time.  Can
> anyone explain what's happening?  Blue

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?
John Dixon                     Lab 44 (223) 334131
Wellcome/CRC Institute         Fax 44 (223) 334134
Dept Genetics
Cambridge University    
United Kingdom       e-m: jpcd0 at mole.bio.cam.ac.uk



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