DNA fragment isolation

bradley turner turner at SPRCORE.BIDMC.HARVARD.EDU
Fri Dec 12 22:43:39 EST 1997


Hi Rachael,

You might have a look at the following references:

Analytical Biochemistry 240, 17-23, 1996
Visualisation of DNA in agarose gels as migrating colored bands:
applications for preparative gels and educational demonstrations
S Adkins and M Burmeister
http://www-personal.umich.edu/~steviema/blueDNA.html
steviema at umich.edu

BioTechniques 21,898-903, 1996 November
Protection of DNA during preparative agarose gel electrophoresis against
damage induced by ultraviolet light
D Grundemann and E Schomig
dirk.gruendemann at urz.uni-heidelberg.de

Also Eric Hugo eric at bcserv.wustl.edu has posted the following to
this news group in the past:

"As a followup to the methylene blue staining discussion
just begun (and from a few months back), I have found that
a slight modification greatly reduces the background
staining problem.  Instead of staining in 0.02% methylene
blue for 30-60 min and then destaining for what seems to
be forever I've found that 0.002% (1/10th X) works just as
well and the background is much lighter.  I have also found
much to my suprise that NuSeive:Agarose (3:1, 4% final) gels
stain very nicely and that dsDNA as small as 75 bp is easily
visualized.  I'm currently using MB staining for all of my
cloning work as I discovered that I get 20-50X as many
positive clones from band isolated DNA when comparing MB
to ethidium bromide/UV transillumination."
----

Comments:

THe visible staining methods avoid DNA exposure to UV altogether
although they tend to be less sensative than EtBr for detecting
DNA.  Thus they are good for isolating large amounts of DNA from
gels.

I tend to use the Gruendemann method more.  It is pretty simple,
adding 1.5mM guanosine or cytidine to the normal TAE electorphoresis
buffer (I also usually add it to the gel and EtBr staining and destaing
solutions-"just in case").  This retains the sensativity of EtBr
staining while presumably protecting the DNA from UV damage.
This reference also specifically evaluates this protective method 
on in vivo transcription-your application.

Hope this helps,
Brad Turner


**************************************************************
  		    Bradley Turner
                Beth Israel Deaconess
                    Medical Center

Harvard Medical School		617-667-1215 phone
Division of Gastroenterology	617-667-2767 fax
Room Dana 536			bsturner at mbcrr.harvard.edu
330 Brookline Avenue		bturner at bidmc.harvard.edu
Boston, MA 02215		turner at sprcore.bidmc.harvard.edu
**************************************************************



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To: methods at net.bio.net
From: "Rachel N. Fish" <rfish at ocf.Berkeley.EDU>
Subject: DNA fragment isolation
Date: Mon, 1 Dec 1997 16:31:17 -0800
Message-Id: <Pine.SOL.3.96.971201162508.22341A-100000 at apocalypse>

Hello-
  I am making DNA templates by PCR amplification to be used in
transcription.  I have two major concerns.  1) The effects of UV on my
fragment.  UV has been shown to have an effect on transcription.  Hence, I
would like to minimize (or do away with!) such exposure.  2) High
efficiency recovery.  Since these templates are a major part of my
research, I would like to make enough to last for awhile.
  After running my PCR samples on an agarose gel, I have tried
electroelution, QIAgen, GeneClean, all with varying (and sometimes very
low) results. 
  Does anyone have any suggestions?  If this question has already been
answered, I apologize for the repeat, but I'm new to this group.  I tried
searching the archives but didn't come up with anything quite appropriate.
  Thank you in advance for your assistance.
  -Rachel Fish
   rfish at ocf.berkeley.edu





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