DNA purification

Dr. S. Bhattacharya sbhattac at crc.ac.uk
Mon Jan 11 06:26:57 EST 1993


Hi all
I got a few requests to explain this method in more detail so am posting it
to the net.

To siliconize glass wool:
Place glass wool in beaker, and about 50 ml of dimethylsilane in another beaker.
Put them both in a vacuum dessicator (in fume hood) and apply vacuum till dessicator
is evacuated. Leave the glass wool to absorb the silicone vapor overnight.

To extract DNA:
1.Cut out band from agarose (1.5% or less in TBE, normal agarose).
2.Briefly blot the agarose block dry.
3.Pierce a hole using a green 21G needle at the bottom of a mini-eppendorf.
  Pack the eppendorf with a small pinch of siliconized glass wool (so that there is about
  4 mm of packed glass wool at the bottom of the tube).
4.remove the cap from a larger Eppendorf and put the mini eppend. into it.
5.Place agarose block containing your DNA into the small Eppendorf
6.Spin 6500 rpm on bench top for 10 min
7. Depending on size of agarose block you should get 50-100ul of aqueous phase
buffer containing your DNA, usually at 10ng/ul, you can check recovery using
UV fluorescence.
8. You can clone or sequence directly; you can also concentrate and remove buffer salts
using an Amicon Microcon of appropriate pore size membrane.

A far cheaper and faster method than various kits on offer.

Best of luck

Shoumo



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