DNA from Polyacrylamide Gels

Ashok Aiyar ashok at meds38956.cwru.edu
Wed Dec 9 19:25:15 EST 1992


In article <1992Dec9.061342.26494 at kudpc.kyoto-u.ac.jp> 
	a52041 at sakura.kudpc.kyoto-u.ac.jp (Michael CHENG) writes:


>  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".

I have had pretty good luck using the "crush and soak" technique for 
fragments that are less that 200 bps (dsDNA).  Recovery was 30% - 40%
I played around with electroelution for a while, but the DNA I recovered 
wouldn't clone into anything - even after a couple re-precipitations 
accompanied by 70% EtOHwashes.

For fragments above 200 bps, I routinely use 2% - 3% agarose gels in 0.5X 
TBE.  While I didn't believe it until I ran my first one of these about a 
year back, I can routinely seperate products that are about 30 - 40 bps
apart in size (eg 250 v/s 280 bps).  I electrophorese them onto DEAE 
Cellulose paper (Whatman DE-81), and elute using high-salt buffer (1.0 M 
NaCl).  Recovery is about 60% - 80% when glycogen is used as a co-
precipitant.

The products have worked in a number of enzymatic reactions such as 
ligations, in vitro transcriptions, ..........

Ashok

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                              Ashok Aiyar 
                      Department of Biochemistry
                       CWRU School of Medicine
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