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

Cotton (or Glass wool) - autoclave for sterilization

3 M Sodium acetate (pH 5.8)

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. Plug small amounts of sterilized cotton into the bottom of 650 ul microfuge tube.

3. Put this 650 ul microfuge tube into a 1.5 ml microfuge tube (collection 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.
  • UV light nicks nucleic acids. Minize UV light exposure.

    5. To elute DNA from agarose gel, centrifuge microfuge tubes at 12,000 rpm for 15 min. at 4oC.

    6. Transfer eluted solution to a new microfuge tube.

    7. Repeat steps 5-6 until all nucleic acids eluted. 3-4x.

    8. To the eluted DNA solution, add 1/10th volume of 3 M Sodium acetate and 20 ug of glycogen.

    9. Extract eluted DNA solutions with Phenol : Chloroform : Isoamylalcohol.

    10. Extract again with Chloroform : Isoamylalcohol.

    11. Add 1 vol. of iPr-OH or 2.5 vol. of 100% ethanol.

    12. Centrifuge to pellet DNA at 12,000 rpm for 30 min. at 4oC.

    13. Remove alcohol.

    14. Wash DNA pellet with 70% Et-OH.

    15. Centrifuge briefly to pull down all liquid.
  • Remove liquid by pipet completely.

    16. Air dry briefly for 5 minutes.

    17. Resuspend DNA pellet in 10-20 ul H2O or TE.



    NOTES



    KIT INFORMATION




    REFERENCES



  • Please send your comment on this protocol to "editor@MolecularInfo.com".

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