DNA purification from polyacrylamide gels / kits?

Harry Harry.Witchel at bristol.ac.uk
Tue Nov 7 08:47:35 EST 1995


In article <9511061736.AA03830 at cockleberry.ncifcrf>, pnh at ncifcrf.govt says...
>
>Timo Hiltunen wrote:
>
>| I am looking for a KIT for DNA purification from polyacrylamide gels.
>| I know the procedure in the Red Book, but does an even easier one 
>| exist? 
>| 
>| All the kits for DNA purification from gels that I've seen 
>| are designed for agarose.
>
>A kit?!??? >:^{
>
>Electroelute it against 6,000-8,000 MW cutoff dialysis tubing in TBE.
>This gives me nearly 100% recovery of 50-200 bp DNA. Build your own
>device or use the eppi-tube technique. References are in here. Grab
>it at my FTP site.
>

Hi --
	I have found crush-elution to be extremely effective for getting DNA 
of ligation quality (size 50-200 bp) out of 8% acrylamide minigels, and crush 
elution is both easier and cheaper than every other technique.  The important 
step is that the acrylamide chip be extremely well macerated.  The steps are

1)  Macerate chip (explained in a moment)
2)  Add 200 ul of TE
3)  Vortex well and, Incubate overnight at 37C
4)  Spin down acrylamide, harvest TE
5)  Add another 200 ul of TE to acrylamide chip, vortex, spin, harvest TE
6)  Pool both samples of TE, and spin hard to get out last of acrylamide
	(ie do not harvest the last 10 ul of TE )
7)  Ethanol precipitate with carrier (40 ul 3M NaOAc, 1 ml EtOH, 1 ul Qwik 
precip or other carrier)
8)  To clean this DNA, one can wash with 70% EtOH and ligate the pellet, but I 
tend to redissolve the pellet in 400 ul TE and reprecipitate (w/o carrier) 
three more times, and finally wash the pellet in 100% ethanol.  This results 
in very clean DNA indeed.

TO MACERATE ACRYLAMIDE CHIP: Prepare in advance a stock of sterilized 0.5 ml 
(ie small) eppendorf tubes which have had their lids cut off and a hole 
punched in the bottom of the tube using a largish gauge syringe needle.  Place 
the acrylamide chip with the DNA into one of the small eppendorf tubes with a 
hole in it, and place the entire little tube inside of a 1.5 ml eppendorf tube 
(ie the normal size).  Balance this assembly in an eppendorf centrifuge, and 
spin -- it may be necessary to remove the top from the big tube as well, but a 
centrifuge with an enclosed rotor (eg Heraeus 13) can have the cap left on the 
big tube.  When you spin the little tube inside the bigger tube, the chip is 
forced down the little tube and crunches its way through the hole in the 
bottom so that it crushes itself as it goes from the little tube into the big 
tube.
	Harry



>@article{Hengen1994Septibs,
>author = "P. N. Hengen",
>title = "Methods and reagents - Recovering {DNA} from agarose gels",
>journal = "Trends in Biochemical Sciences",
>volume = "19",
>number = "9",
>pages = "388-389",
>month = "sep",
>year = "1994"}
>
>-Paul.
>
>*****************************************************************************
**
>* Paul N. Hengen, Ph.D.                           
/--------------------------/*
>* National Cancer Institute                       |Internet: pnh at ncifcrf.gov 
|*
>* Laboratory of Mathematical Biology              |   Phone: (301) 846-5581  
|*
>* Frederick Cancer Research and Development Center|     FAX: (301) 846-5598  
|*
>* Frederick, Maryland 21702-1201 USA              
/--------------------------/*
>* - - - - - Methods FAQ list -> http://www-lmmb.ncifcrf.gov/~pnh/ - - - - - - 
*
>* - - -  Anonymous FTP from ftp.ncifcrf.gov as file pub/methods/FAQlist - - - 
*
>*****************************************************************************
**




More information about the Methods mailing list