IUBio

DNA isolation from agarose gels

Hiranya S. Roychowdhury hroychow at NMSU.EDU
Tue Mar 9 13:47:25 EST 1999


A lot of people queried about the DNA insert extraction protocol. There
have already been replies from others outlining it, but some seem to have
missed those postings. So, here it is:

1. Stuff the bottom of a 0.5mL microfuge tube with siliconized glass wool,
making a tight filter wad.

2. Puncture the bottom with a 18-20gauge needle, autoclave and store for
future use. 
I have my students prepare several of these "columns" at a time.

3. After separating out the fragment/plasmid of interest on the agarose gel,
visualizing and documenting it, cut the target band out with a razor blade
and place it in the "column". 

4. Freeze the column with the gel slice. Use either the -70 C freezer or
liq.N2. Dry ice/alc bath is OK too, but the writings on the tube has to be
with a marker that will not be washed off (VWR pens?).
        This freezing step is dispensible, but the yield is reduced to about
80% if done w/o freezing. So, if the slice contains a huge amount of DNA
(ie. more than is needed for your use, eg., ligation, transformation etc.),
this step may be skipped.

5. Place the "column" with the gel slice in a 2mL or 1.5mL microfuge tube
and centrifuge at 10K rpm for 2min.

5. Remove the the eluate in a separate tube. Add 100uL of TE to the column
and spin again as before, to wash. A longer spin may be used to recover most
of the remaining DNA.
I should mention here that larger the DNA, longer the spin required. So, it
is better to determine that impirically. Also, the speed may be varied. 

6. Pool the eluate  with the first, extract 2x with TE or water saturated
n-butanol,
add 1/10 vol of 3M KOAc or 7.5M Amm-Ac to the aq. phase, and ppt the DNA
with EtOH (2vol) or isopropanol (1 vol).



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