Reamplifying PCR bands
Terrence P. Delaney
tpd4 at cornell.edu
Fri May 9 17:58:28 EST 1997
In article <5kt3oa$fsi at vixen.cso.uiuc.edu>, dneece at ux1.cso.uiuc.edu (David
> In article <336A131E.3B3C at exeter.ac.uk>, mabenson at exeter.ac.uk says...
> >Has anyone any experience of PCRing up bands that have been run on
> >agarose gels without actually removing the agarose?
> >Any ideas would be gratefully received!
> I've used the following method on 1% agarose/TBE gels with success:
> 1. Cut out the band of interest and put it into a 1.5ml tube.
> 2. Freeze the gel slice (in the tube) @ -20, then thaw @ room temp.
> 3. Repeat step two for a total of 3 times freeze/thaw.
> At this point, you will notice liquid along with the agarose
> slice in the tube -- I'm assuming that freezing forces water
> [containing DNA] out of the gel slice.
> 4. Spin the tube @ 10,000 rpm for a few seconds.
> 5. Pipet off the supernatant and dilute 1/10.
> 6. Use 1 ul of dilution for PCR.
> To speed things up, I usually perform the freeze/thaws in the
> -80 freezer (5-10 min) and 37oC water bath (5 min). This may
> promote some shearing of the DNA, but it has worked well for me...
> Good luck,
> David Neece
> dneece at uiuc.edu
We simply excise the band from a low-melt gel, dilute it substantially
(>100-fold) with water and use a ul or so in a PCR reaction. It works
great. Nested primers are best if you want to ensure that you are
re-amping the expected species (not usually a problem, though).
Terrence P. Delaney, tpd4 at cornell.edu (607) 255-7856 (fax-4471)
Cornell University, Department of Plant Pathology
341 Plant Science Bldg, Ithaca, NY 14853
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