Molecular Techniques and Methods
Elution of DNA From Agarose Gel by Squeezing
Copy Right © 2001/ Institute of Molecular Development LLC
INTRODUCTION
MATERIALS AND SOLUTIONS
650 ul microfuge tube
1.5 ml microfuge tube
21 gauge needle
Glass fiber (or Glass wool) - autoclave for sterilization
3 M Sodium acetate
(pH 7.0)
10 ug/ul Glycogen
Phenol : Chloroform : Isoamylalcohol
(25: 24: 1) (pH 6)
Chloroform : Isoamylalcohol
(24: 1) (pH 6)
TE (pH 8)
PROCEDURES
1. Prepare a 650 ul microfuge tube which is poked at the bottom with a 21 gauge needle.
2. Put small amounts of sterilized glass fiber (or cotton) into the bottom of 650 ul microfuge tube.
3. Put this 650 ul microfuge tube into a 1.5 ml microfuge tube.
4. Cut desired DNA band out of the agarose gel (0.6%) and place in a 650 ul microfuge tube prepared from step 3.
5. To elute DNA from agarose gel, centrifuge microfuge tubes at 12,000 rpm for 15-20 min. at 4
o
C.
6. To the eluted DNA solution, add 1/10th volume of
3 M Sodium acetate
and 40 ug of
glycogen
.
7. Extract eluted DNA solutions with
Phenol : Chloroform : Isoamylalcohol
.
8. Extract again with
Chloroform : Isoamylalcohol
.
9. Add 2.5 vol. of 100% ethanol.
10. Centrifuge to pellet DNA at 12,000 rpm for 20 min. at 4
o
C.
11. Remove ethanol.
12. Centrifuge briefly to pull down all liquid. Wipe out residual liquid by Kimwipe. Becareful, not to touch the DNA pellet.
13. Air dry briefly (for 1-2 minutes at the bench top). Resuspend pellet in 10 ul TE.
NOTES
Do not use agarose gels higher than 0.6%. DNA will not be eluted in higher % gels (>0.6%).
In step 5, it is not necessary to add any elution solution, since agarose gel has enough solution to elute DNA.
In step 10, it is not necessary to keep the eluted DNA solution in a freezer for precipitation.
In step 13, do not over dry the DNA pellet. Otherwise, it will be very difficult to resuspend the DNA pellet in TE.
KIT INFORMATION
REFERENCES
Please send your comment on this protocol to "
editor@MolecularInfo.com
".
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