Desalting of Plasmid DNA after Cesium Preps

Chris Boyd chrisb at hgu.mrc.ac.uk
Mon Jul 13 04:25:38 EST 1998


Frank O. Fackelmayer (fof1 at chclu.chemie.uni-konstanz.de) wrote:
: Chris Boyd wrote:
: > 
: -snip 

: > I think you always lose a lot doing ethanol precipitation, particularly
: > in the washing stage which is necessary to remove salt traces.
: > 
: > You have to make a choice: time or yield + quality.

: I disagree. Some years back we did a parallel test to see what is better,
: direct EtOH precipitation after dilution (see my first posting for the
: method), or dialysis. Result: yield with direct precip was *TWICE* that of the
: dialysed sample (3mg compared to 1.6mg). In addition, direct precip was
: finished in 2h, while dialysis took 5h plus overnight plus EtOH precip (1h).
: That´s 2:16, factor 8 in speed. Not bad for a method with twice the yield. I
: have to add that we did not see ANY difference in DNA quality (restriction
: digests, sequencing, transfection were all exactly the same). So, slower is
: not always better.

Frank, I've no doubt your results are valid, but why do you EtOH ppt
AFTER dialysis? I suspect this is where you're losing a lot of DNA:
after all, it can't get out through the dialysis membrane. Also, it's
not surprising you see no difference in quality if in effect you tested
dialysis simply as an additional intervening step before precipitation.
The differential you observed can perhaps be accounted for by the
precipitation in the "no dialysis" method being more efficient due to
(?) higher initial concentration of DNA and/or differing salt
environment (CsCl + NaCl as opposed to NaAc). For medium to high-copy
number plasmids, I find there is no need to concentrate DNA after
dialysis -- you will have 0.1-1 ug/ul in the dialysate which is enough
for most purposes. I've always been puzzled as to why others like to
have plasmid DNA stocks at 5-10 ug/ul which means pipetting out tenths
of microlitres for digestions etc.

BTW, I notice in your earlier reply you extract EtBr with
CsCl/isopropanol, then with NaCl/isopropanol. I extract with
NaCl/isopropanol (by which I mean water + isopropanol + NaCl in such
proportions that (i) there is solid undissolved NaCl at the bottom and
(ii) a visible boundary between the upper alcohol and lower water
phases) all the way, with no problems. It saves on expensive and
harmful CsCl.

: BTW, I enjoy this thread, as it shows there are still other people out there
: who prefer good old CsCl over the kits used in almost all labs. Its simply
: better, that´s why.

Agreed, but have you ever tried to persuade Qiagen reps of this point
of view?

Best wishes,
--
Chris Boyd                      | from, but not \ MRC Human Genetics Unit,
Christopher.Boyd at hgu.mrc.ac.uk  | on behalf of  /  Western General Hospital,
http://www.hgu.mrc.ac.uk/Users/Christopher.Boyd \   Edinburgh, EH4 2XU, SCOTLAND



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