isolating 197bp fragment

SURESH KUMAR K.G. skg at biochem.iisc.ernet.in
Wed Nov 22 16:30:06 EST 2000



Hello,

You can use the glass wool elution method.
I think a protocol for this has been posted earlier in the group.
Any way here it goes..

1.Pierce a small 0.5ml eppendorf with a needle (18g) and plug it with
  silianized glass wool. 

2.Keep it in a large eppendorf and spin at 6500 rpm for 5min 

3.Cut the gel(1.0%)slice into very small pieces.

4.Put it back into the eppendorf with glass wool and place it in a fresh
  large tube.

5.Spin at 6500 rpm for 20 min.Collect the eluate. 

6.Take an aliquote, check in a gel. Use an aliquote directly for
  labelling,ligation etc..

A few tips...


Start your digesion with large amounts of plasmid so that  you can
visuallize the fragment with least amount of Ethidium bromide, for
cutting. 

Run the gel in a clean tank with fresh 1X TAE buffer.

Cut out the band with precision so as to reduce the volume of gel.


Hope this helps

Happy probe making....

Regards

Suresh Kumar K.G.
Dept of Biochemistry 
IISc, Bangalore



 

On 22 Nov 2000, Robert Mihalek wrote:

> I need to isolate a 197bp dsDNA fragment in sufficient quantities for
> making a radioactive probe. I have the fragment subcloned in a plasmid
> and can easily isolate it from the plasmid with an EcoR I digest.
> 
> Most kits for gel purifying dsDNA seem to have a cut-off at around
> 200bp. I'm wondering if anyone has some tips on how to best proceed
> with isolating this fragment? 
> 
> _______________________
> Bob Mihalek  rmihalek at FORMULA1.partners.org
> remove name of race series to reply
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