holger.neumann at ruhr-uni-bochum.de
Wed Feb 24 17:09:13 EST 1999
Hi amherrera (?!),
> Hi netters!
> I am trying to cut a plasmid first with Kpn I and with EcoR V in a
> second step.
> The vector linearized well after Kpn I. After these all following
> steps (1:1 phenol+cloroform, 2-butanol, 0.5 vol NH4Ac 0.6 vol
> isopropanol, ethanol 70%), I proceed cutting with EcoRV and
> the vector did not give the expected band.
> Surprisingly the same vector is diggested with EcoRV if I start
> with this enzyme first.This never happpened to me before.
> Where's the problem?
I am a M.D. student and got a lot of knowledge while working on my
doctoral thesis, which are not finished :-(
I made the same experience as you explained with the enzymes EcoRI and
NotI -which are standard enzymes in molecular biology- when restricting a
comercially distributed plasmid. I tried several ways to solve the
problem and the results presented as your's: both enzymes function well
in the first step, but don't in the second one and unfortunately the
buffer conditions for both enzymes differ as maximal as possible which
makes double-digest unpossible !!
After several days I tried to change the method of purification after the
first step. And luckily that was the solution to my problem: formerly I
tried to purify DNA using silica beads purchased from Merck, which made
good results with other plasmids but failed with this one (don't know what
the explanation to this is, but all other researchers who tried to perform
failed as I did !!), alternately I tried ethanol/NH4Ac precipitation and
the High-Pure-Plasmid-Isolation-System purchased by Boehringer. Using
these methods for purification made it possible to perform the second
I doubt this would help you much because you already used
ethanol/NH4Acprecipitation, but I wrote to present this curious case to
all netters here !!
M.D. student Holger Neumann
University of Bochum
endocrinology lab at university clinic 'Bergamnnsheil'
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