Microwave restriction digests

Charlie Wright Genetics cw117 at mole.bio.cam.ac.uk
Tue Sep 27 08:18:43 EST 1994


jpcd0 at mole.bio.cam.ac.uk (John Dixon) writes:

>In article <Prenom.Nom-220994100709 at agrobacterium.rsvs.ulaval.ca>,
>Prenom.Nom at rsvs.ulaval.ca (Prenom Nom) wrote:

>> In article <jpcd0-2009942104500001 at macr1-1.welc.cam.ac.uk>,
>> jpcd0 at mole.bio.cam.ac.uk (John Dixon) wrote:
>> 
>> > 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.
>> 

>Excluding the bottom one (which I reported) these two both leave the DNA
>digesting for eight minutes minimum at room temp. This is quite a long
>time if you are using 20 units of enzyme (1u cuts 1ug completely in 60 min
>@37C, 20u may well cut 1ug in 3 minutes, assuming a linear relationship
>and a nice warm room).

>Enzyme assays are frequently done on lambda DNA, and according to
>Stratagene single sites on plasmid DNA give a unit assay 6-10 times
>higher. 

>If you have ever accidently added restriction enzymes to a sample and
>regretted it as I have once or twice, you will have discovered that even
>with almost instant addition of EDTA and heat inactivation the sample is
>already cut. Perhaps this is just Sod's Law operating - I've never tried
>the control of adding the enzyme I meant to and then tried to inactivate
>it :-).

>I suspect that if you tried the controls, you would find them digested too
>(probably better as some enzyme may well be inactivated each time you
>microwave the digest).

>Your report that this m/wave protocol works on 'total' (genomic?) DNA is
>much more interesting - could you post some more details on units, ugs,
>what organism, etc

>Thanks

>-- 
>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

We fiddled with this in our lab a few months ago, and were puzzled but 
unimpressed.  We found that with EcoRI, the microwaved tube, room 
temperature tube, and 37 degree tube (all for five minutes) were 
virtually indistiguishable from one another on a gel.  All cut to completion 
(plasmid) and the microwave showed a little degradation).

Using BamHI (or HindIII, I forget) we found that 37 produced a partial, 
room temperature showed little cutting, and the microwave made a hash of 
the DNA (just a smear on the gel).

Although I'm very skeptical of the technique, no one could explain why a 
20-50 micorlitre sample didn't just evaporate to nothing in a microwave 
on high for five minutes!  We tried water first (we had a little 
sweepstakes on what would happen) and it didn't even notice the 
microwaves... odd.  Guesses ranged from "sample smaller than the 
wavelength of the microwaves" to "eppendorf tubes are microwave-proof" 
and "dunno... probably elves".

Any other results with enzymes or explanations of the boiling question 
would be intriguing.

Cheers,
Charlie

-- 
C. R. Wright                                    Dept. of Genetics
+44 (0)223 333970 telephone                     Univ. of Cambridge
+44 (0)223 333992 telefax                       Downing Street, Cambs.
cw117 at mole.bio.cam.ac.uk                        CB2 3EH, England



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