As good a place as any to record the "freeze-squeeze" method of DNA extraction from gels:
1. Run gel as usual
2. Isolate band using long-wave UV
3. Freeze lump of agarose containing band of interest (upwards of 20 minutes)
4. Place agarose lump in top of SpinX-type spin column, and spin 2 minutes
5. Mush remaining agarose with 200 µl TE and spin 5 minutes
6. Phenol-chloroform extract the flowthrough (use 1:1 volumes)
7. Ethanol precipitate.
8. Resuspend in appropriate volume of water/whatever buffer.
Note to self: Do not freeze in the SpinX column. This can make a mess of the centrifuge.
If this means nothing at all to you, you are probably better off not knowing.
Comments
What is the advantage of freezing, except that it rhymes with squeezing? We just mush and sometimes do the ten-minutes-at-50° thing; it works alright, too.
Posted by: Alethea | March 5, 2007 08:27 PM
Historical reasons.
If it works without freezing, then that's even better. I shall do the experiment this week.
Posted by: BK | March 5, 2007 08:58 PM
Can you omit the column entirely? Or does the agarose make too much guck in the PCl2 extraction?
Just wondering. I'm a column whore myself, or I was.
Posted by: Whiffling in a columnar fashion | March 8, 2007 05:43 AM
I had always understood that freezing helped to weaken the gel so that you got better mushing and release of the DNA. Pretty convinced that just PCI will be messy — you'd probably have to re-extract, which is tedious.
Posted by: BK | March 8, 2007 10:47 AM