PCR from dried sequencing gels

pattonaj%phvax.dnet at sb.com pattonaj%phvax.dnet at sb.com
Mon Nov 14 17:40:54 EST 1994


Yes it is possible to perform PCR on bands excised from dried sequencing
gels, you have to do it in differential display. Here's the method I use,
it works most of the time.
Run denaturing gel. Don't fix. Dry onto Whatman 3M. Autorad.
Reorientate film with gel. Cut out desired band(s) from gel/whatman using
razor. I usually then put another bit of film on gel and re-autorad to make
sure that I've cut out the right band.
Soak gel/whatman slice in 100 microl dH2O for 10 mins.
Boil the tube for 15 min. Seal cap well with lidlock or parafilm.
Spin in microcentrifuge 2 mins.
Transfer supernatant to new microfuge tube. Add 10 microl 3M sodium acetate,
5 microl glycogen (10mg/ml) and 450 microl 100% EtOH. Ppt at -80 C 30 mins.
Spin 10 mins at 4 C. Wash in 85% EtOH.
Dissolve pellet in 10 microl dH2O. Use 4 microl for rePCR, 40 microl reaction
volume 20 microM dNTP.
I run 30 microl of the rePCR on an agarose gel. You should see a band. If not
use 4 microl of a 1:100 dilution of the first round PCR as a templatefor anotherround of amplification.
Oh should have mentioned that I do 40 cycles.

Hope this helps

Amanda Patton



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