Help recovering 200bp DNA fragment

mike roper you at somehost.HSC.Colorado.edu
Wed Aug 16 15:24:26 EST 1995


In article <95216.110837VHWBC at CUNYVM.CUNY.EDU>, Harshinee Wijesinghe <VHWBC at CUNYVM.CUNY.EDU> says:
>
> 
>I have been trying to elute and recover a 200bp insert (cut from a plasmid)
>from agarose gel without much success. I definitely know that the DNA came
>out of the gel as I monitored it under UV light and subsequently stained



<<snip>>

I _never_ had much luck using this method.  The DNA is far too dilute and
easily lost on the side of the tube.  Here's a method which works
spectacularly:

1.  Obtain some DEAE paper, specifically Schleicher and Schuell NA45.
2.  Run the electrophoresis in the buffer of your choice (i.e. TBE).
3.  Take the gel out of the box and cut out a small block _IN LINE WITH
    THE BAND_.        band      ____
                     --------- |____  <<--block
4.  Slide the block back and insert a small strip of NA45 paper into the
    space.
5.  Close the space (i.e. slide the block back into place).
6.  Turn the gel 90 degrees and electrophorese the band _END ON_ onto
    the paper.  This will result in a tight streak ca. 2 X 2 mm on the 
    paper.
7.  Remove the paper and wash it 2 times in distilled or deionized water.
    (15-50 ml per wash).
8.  Trim the streak using the transilluminator to reveal the ethidium-
    stained DNA.  _Do not_ over illuminate as you can crosslink the 
    DNA to the paper.  Trim all of the non-loaded paper away from the 
    DNA in the streak.
9.  Place the _small_ strip of paper into a microcentrifuge tube, and 
    cover it with a minimal amount of 1 M NaCl. This should be about
    50-100 microliters.
10. Heat to 65 degrees for 15-30 min (until the DNA emigrates from the
    paper).
11. Transfer the solution into a second microcentrifuge tube and add 2
    volumes of ethanol.  IMMEDIATELY spin the DNA down at 13,000 X G/5 min.
12. Wash 2 times with 70% ethanol and redissolve in T.E.
13. Test-run the DNA to estimate recovery.  Recoveries are typically
    75% or greater with this method.

Good luck

Mike



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