DNA from Polyacrylamide & Protein Fusion and Purification System

[Victor Jongeneel]

Basavaraju Shankarappa (bsh at MED.PITT.EDU) wrote:
: >   Hi! I was wondering if anyone out there knows of any company that sells a kit
: > for extracting DNA from polyacrylamide gels. According to Molecular Cloning
: > (2nd Edition) by Sambrook et al on page 6.46, there exists a "Crush and Soak"
: > method. However, the method is lengthy and inefficient. With so many kits
: > available for extracting DNA out of agarose gels, I haven't been able to find
: > one that's for use with polyacrylamide gels. Unfortunately, I must run my DNA
: > samples on PAGE. I would appreciate it if anyone could provide any info. or
: > even suggest a better method other than "Cursh and Soak".
: > Michael Cheng
: > Dept. of Biological Chemistry
: > Kyoto University
: > a52041 at sakura.kudpc.kyoto-u.ac.jp

: There is a system of solubilizable acrylamide gels for isolation of DNA
: fragments by Dr. Norman Hansen.  He was our teacher and he was a great
: one.
: 	In this system, the gels are crosslinked by disulfide bridges and
: the gels are supposed to be quite effective in resolving MW ranges from
: 21 kb down.  I do not know whether anybody markets this system.  You can
: obtain the disulfide containing bis from Bio-Rad.  The gels are solubilized
: by treating with mercaptoethanol and the DNA purified by DEAE cellulose. 
: I guess you should be able to clean DNA with other methods also.
: 	The reference is J.N. Hansen.  Use of solubilizable acrylamide 
: disulfide gels for isolation of DNA fragments suitable for sequence
: analysis.  Anal. Biochem. 116: 146-151 (1981).

: Raj Shankarappa
: bsh at med.pitt.edu

Michael,

Another (and easier) way is to electro-elute your DNA from the gel.
There are several commercial systems available to do this.  We have
tried the BioTrap from Schleicher & Schuell (may be sold under
different trade names outside Europe) and a "salt trap" model sold by
IBI/Kodak.  They both work fine, but I prefer the IBI system, which is
quicker and allows you to elute up to 6 samples simultaneously.  All
you have to do after elution is EtOH precipitation.

Hope this helps.  Good luck!

Victor

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C. Victor JONGENEEL                       vjongene at isrecmail.unil.ch
Ludwig Institute for Cancer Research, Lausanne Branch
Chemin des Boveresses 155                       FAX: +41-21-653-4474
CH-1066 EPALINGES				Tel: +41-21-653-6275
Switzerland
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