DNA purification

GIETZ at bldghsc.lan1.umanitoba.ca GIETZ at bldghsc.lan1.umanitoba.ca
Mon Jan 11 14:34:00 EST 1993


%%%%%%%%%%%%%Stephanie Writes %%%%%%%%%%%%%
%In article <1993Jan11.112657.23798 at crc.ac.uk>
%sbhattac at crc.ac.uk (Dr. S. Bhattacharya) writes:
 
>
>Hi all
>I got a few requests to explain this method in more detail so am posting it
>to the net.
>
(Stuff Deleted)
>
>A far cheaper and faster method than various kits on offer.
>
>Best of luck
>
>Shoumo

% I used this method for a while after hearing about it from a friend.  It
% seemed to give me decent recovery of DNA.  However, I could never use
% DNA purified in this way for ligations.  I just would not ligate at all.
% Any ideas?
 
% Stephanie
 %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
Hi Stephanie
I have have the same problems with DNA purified this way. 
To get around this problem I
purify DNA fragments from Agarose using the Method of
Girvitz et al (1980) Anal.Biochem. 106:492 as described 
in Maniatis, Fritsch and Sambrook (1982) pg 168.
This method inserts a small piece of dialysis membrane fronted 
with 3MM whatman paper (to hold the drop of buffer to the 
membrane when you remove it) into a slit cut in the gel just in
front of your band of interest . you then
 catch your DNA frag  on the Whatman/membrane 
by electrophoresing it  in the correct
direction.  Its an old method 
but works very well giving high yields of DNA that one can do  
 anything with including ligations. There is no need for special
agarose etc... I always do blunt end ligations
with DNA purified this way with no problems.
A few hints:
    1. I use Whatman 3MM paper not DEAE cellulose paper 
        for better yields.
    2.  Avoid spinning your elutions too hard.  This dries out 
        the paper and reduces your yield substantially.  I spin at 
        2500 rpm for 
        the first two spins and then spin hard the final one. Most 
        your DNA comes off on the 1st and 2nd elution.
    3. I add the first elution wash before spinning the paper dry.
        This seems to increase yield.
    4. An overnight incubation at -20 seems to give better EtOH pellets
       ( A 1X g sedimentation) Other wise your pellet is spread over the
        entire outer face of the tube (still recoverable).
    5.  I only do one phenol:choroform extraction.
    6. Use long wave UV to visualize your bands (this is important 
        especially for ligations.)

I have even purified 10 - 12 Kb MboI yeast DNA to make libraries 
with great success.  

If anyone would like more details just drop me a note.

Dan Gietz
U of Manitoba
GIETZ at BLDGHSC.LAN1.UMANITOBA.CA
        



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