DNA purification from agarose gels

CMMID Va Tech dna at mail.vt.edu
Fri Jul 14 09:25:25 EST 1995


Subject: Re: DNA purification from agarose gels
From: xunfang
Date: 3 Jul 95 22:35:58 EDT
In article <1995Jul3.223558.14926 at wvnvms> , xunfang at wvnvms.wvnet.edu
writes:
>In article <3sp9rk$1tp at lyra.csx.cam.ac.uk>, rw200 at cus.cam.ac.uk (R.
Woodward) writes:
>> Dear all
>> 	I would like to canvas opinion on the best kit or perhaps method to
use
>> 	when purifying DNA fragments from agarose gels, at the moment we use
the
>> 	'EluQuik' kit from S&S the yield is good and so is the purity but its
>> 	alittle fiddly especially if you perform alot of isolations at one
time
>> 	. I want to use the purified fragments in ligation reactions.
>> 	Thanks to all Robert
>> 
>> 	R.Woodward
>> 	Dept Pharmacology
>> 	Tennis Court Rd
>> 	University of Cambridge
>> 	Email rw200 at cus.cam.ac.uk
>
>Try Qiaquick Gel Extraction Kit from Qiagen. It takes only 20 minutes for
>one sample. The recovery is around 50% or more.

Subject: Re: DNA purification from agarose gels
From: Tim, tkuwada at neuron.uchc.edu
Date: 13 Jul 1995 20:27:06 GMT
In article <3u3viq$9gl at threed.uchc.edu> Tim, tkuwada at neuron.uchc.edu
writes:
>In article <bio.tamu.edu-2906951005260001 at ricelab-centris650.tamu.edu>
>bio.tamu.edu writes:
>>I am interested in what other people think about Qiagens new Qiaquick
>>columns compared to Promega's wizard PCR preps.  Has anybody done a
direct
>>comparison of these two kits for purification of DNA from gels?
>
>We have not used the Qiagen kit but have had poor yields with the
>Promega kit.
>When we have attempted to purify plasmids (>3kb) with the Promega kit
>we have consistently
>recovered a 1.5kb.  All of our reagents have been filtered, thus it is
>unlikely 
>that this a contaminating band.  To date we have 0% yield of the
>desired DNA.  
>and
>I would be interested in hearing from anyone else who has had similar
>problems.   
>
>t.kuwada at neuron.uchc.edu

Tim, 
Hi, we've combined the promega protocol with the BioRad prep a gene
matrix using the promega columns and get 75% to 100% recovery from 200bp
to 15 kb in 10 min.  Seems unbelievable but about ten people here have
been able to do this.  Try this procedure:
1.Melt gel slice
2.add 3 vol BioRad binding buffer 
3. add 3 times recommended amount of matrix (vortexed) for in gel.   (Use
recommended amount for direct purification.)
4. vortex quickly.
5. set for 1 min
6. put into syringe connected to promega wizard column
7. push through slowly. (1 drop/ 3 sec)
8. disconnect column from syringe
9. pull out plunger and reconnect
10. add 90-95% isopropanol (2ml)
11. push through at same rate 
12. spin 12,000 g in 1.5 ml tube for 2min
13. dry in 68 C hybridization oven or in a speed vac dryer for five in
(***important to remove residual isoprop.)
14. add 30-50ul of water or TE and let sit for approx. 1 min.
15. spin again 2 min 12,000 g
16. run DNA (in tube) on gel to check conc.

This procedure works great in our building.  Check it out.

Good Luck!!!

John R. McQuiston



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