Transcript DNA ISOLATION FROM AGAROSE GELS WITH DEAE PAPER

DNA ISOLATION FROM AGAROSE
GELS WITH DEAE PAPER
Kris-itd.unair.ac.id (for education purpose only)
MATERIALS:
The DEAE paper can be used as supplied by Schleicher and Schuell,
however the binding capacity can be increased by washing the
paper in 10mM EDTA, pH 7.5 for 10 minutes, 0.5M NaOH for 5
minutes, followed by several rinses in distilled water. The paper can
be stored at 4oC for several months.
NET buffer:
0.15M NaCl
0.1mM EDTA
20mM Tris-HCl, pH 8.0
High salt NET buffer:
1.0M NaCl
0.1mM EDTA
20mM Tris-HCl, pH 8.0
METHOD:
After the DNA bands have been separated electrophoretically (at around
80 V), the gel is viewed under long wave UV light and a slit cut in the gel,
just ahead of the desired band. A piece of DEAE paper is inserted in the
slit. Another piece of DEAE paper can be inserted behind the desired
band, to prevent unwanted bands being trapped by the paper.
Electrophoresis is continued for approximately 10 to 15 minutes with the
voltage increased to 100-120 V.
The DEAE paper is removed and binding of the DNA checked by viewing
the paper with long wave UV light. The paper is then placed in an
eppendorf tube containing 500 µl NET buffer to wash away remaining
agarose. The bound DNA can now be stored at 4oC for several days. The
DNA should always remain covered with buffer to prevent irreversible
binding which may occur if the paper is allowed to dry out.
To elute the bound DNA, place the DEAE paper in an eppendorf tube
containing 250 µl of high salt NET buffer so that the paper is completely
covered. Make sure that the side of the paper containing the DNA faces
the buffer and not the side of the tube. If necessary, centrifuge briefly to
submerge the paper.
Incubate the tube at 55-68oC for 45 minutes with occassional swirling (or
until all the DNA has been eluted as checked under long wave UV light).
Remove the buffer to a fresh tube and add 50 µl high salt NET buffer to
wash the membrane. Remove the additional buffer and add to the fresh
tube.
Centrifuge the buffer for 30 seconds to pellet any remaining DEAE paper
fibres and remove the buffer to a fresh tube (leaving approx. 20 µl in the
bottom of the tube).
Extract the residual ethidium bromide with 3 volumes of water saturated
butanol.
Remove and retain the bottom layer and add 1/20 volume of 3.0M sodium
acetate, pH 5.25 and 2.5 volumes of cold absolute ethanol. Incubate at 70oC for at least one hour. Ethanol precipitate and vacuum dry.