Dennis J. Templeton
djt2 at po.CWRU.Edu
Fri Oct 2 16:31:55 EST 1992
In a previous article, KNECHT at UCONNVM.UCONN.EDU () says:
>Hi Netters- I remember reading somewhere that you can use ethidium bromide in
>restriction digestions to make partial digests. Does anyone know a protocol or
> reference to this technique. Thanks- Dave Knecht (KNecht at uconnvm.edu)
The earliest reference I have for this is Parker, Waton, and Vinograd, PNAS
74 (1977) pp851-855. We have used it ir a variant several times.
Two comments: It works well only for *some* enzymes. They used HindIII
and HpaI, I have confirmed that it works very well for HindIII. Other
enzymes are slowed, but not fully inhibited after the first cut.
Also, these workers (and I) used empirical tests to determine the optimal
concentrations of EtBr and enzyme. A typical reaction is 1ug plasmid
+5-20ug EtBr + 2-10 U enzyme in 20 ul. The Parker paper also varied the
temp from 4 to 55 degrees, and said that higher was better.
You *never* get exclusively full length linears, but EtBr does help getting
linears over just limiting enzyme.
For routine partials, we just do serial dilutions of enzyme into DNA
(plasmid is cheap) and run all the products out on a long gel with good
markers. Usually somewhere between 0.5u/ug and 100x less gives reasonable
By the way, it is MUCH easier to use partial digests if you cut fully with
one enzyme, then do partial digests (by dilution) with the second enzyme.
That way (since you know the location of the first cut) you can identify
the appropriate partial with ease, AND since the two ends are different you
can force clone it into vector cut with two enzymes. Fully cut
contaminating DNA won't interfere, since it won't (with two similar ends)
ligate into the twice cut vector.
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