ISOLATION OF DNA FROM AGAROSE GELS WITH DEAE PAPER
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ISOLATION OF DNA FROM AGAROSE GELS WITH DEAE PAPER
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 4o C 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 80v), 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 100-120V.
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 500ul NET buffer to wash away remaining agarose. The bound DNA can now be stored at 4o C 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 250ul 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 55-68o C 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 50ul 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 ul 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 -70o C for at least one hour. Ethanol precipitate and vacuum dry.
