purifying dna from gel slices

vilimf01 at mcrcr6.med.nyu.edu vilimf01 at mcrcr6.med.nyu.edu
Sun Nov 24 23:08:19 EST 1996


In article <56iici$51r5 at ncisun1-nf0.ncifcrf.gov>, pnh at ncifcrf.gov (Paul N Hengen) writes:
> Kirk A. Bartholomew (kirk at NODULE.MCGILL.CA) wrote:
> 
>> I have used the syringe/ 2 micron filter  squeeze method many times with
>> good success.  However I recently began using a 0.2 micron filter setup
>> in a microfuge tube manufactured by Amicon.  This method works as well
>> or better than the syringe filter and the price/extraction is cheaper
> ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~^^^^^^^???
>> than the syringe filter for small amounts of agarose (around 50 mm
>> cubed).  In my hands the procedure requires longer spin times than
> ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~^^^^^^^^^^^^^^^^^
>> recommended by the manufacturer but that is my only deviation from the
>> recommended procedure.  This procedure requires ETOH precipitation for
> ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~^^^^^^^^^^^^^^^^^^^^^^^^^^^
>> concentration or the use of another size separation device manufactured
>> by amicon called a microcon microconcentrator.  These microconcentrators
> ~~~~~~~~~~~~~~~~~~~~~^^^^^^^^^^^^^^^^^^^^^^^^^^
>> come in a variety of molecular weight cutoffs and are almost 100%
>> efficient in concentrating  and removing salts etc. from DNA samples.  I
>> am not a sales rep of Amicon and have no connection with the company
>> except as a very pleased customer.
>> good luck
>> 
>> kirk
> 
> You forgot to mention the cost of the microconcentrators. These are far
> more expensive than syringes, both in time and money. I think I'll stick
> with the syringe technique.

I'm not sure I understand, not having used the filter technique
myself.  If you mean the 0.22um, my fisher catalog lists them as $98
for 50 or 2$ a piece.  0.2um spin filters in the same catalog run
$81.50 for 50 or 1.63 a piece.  Not much of a price difference.  I
personally love the squeeze technique (freeze decreases yields in my
hands).  As a matter of fact, I am throwing away my Qiaex II.  I run
my gel (0.5x TBE) cut out my bands with minimal excess agarose, spin
for 10min, and ligate 4ul insert, 4ul vector (also spin purified) 2ul
BRL ligase buffer, 1ul BRL ligase.  Leave on my bench for 1-3hr,
transform and there you go.  It has worked for me on a number of
occasions.  I havent had the nerve to try the paper slurry filter
(biotechniques 17(4) p634 1994) since the spin filters have been
working straight in my ligations and I'm worried that something in the
paper may interfere in my ligations.  Maybe when I have more time,
I'll play around with that (talk about cheap!).

	The other thing that I've just tried is the quick
transformation protocol described in the recent Fermentas Newsletter
and in NAR 24(3) p536 1996.  Basically, you mix the ligation or DNA
with the competent cells, mix, leave on ice 5min, and plate onto
prewarmed plates.  This has worked for me the one time I tried it and
its a real timesaver.  Be aware that there are cell types that don't
like this protocol, and it doesn't work so well for kanamycin
resistance (but works fine for amp,tet, cam, DH5alpha, and XL1 blues).

				Regards			SVEN


> 
> --
> *******************************************************************************
> * Paul N. Hengen, Ph.D.                           /--------------------------/*
> * National Cancer Institute                       |Internet: pnh at ncifcrf.gov |*
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