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Method: Isolation of Restriction Fragments from Agarose Gels by Collection onto DEAE Cellulose

4/18/1990

C. Helms


Principle:

% agarose in the gel Efficient range of separation of linear DNA molecules (kb)
0.3 60 - 5
0.6 20 - 1
0.7 10 - 0.8
0.9 7 - 0.5
1.2 6 - 0.4
1.5 4 - 0.2
2.0 3 - 0.1

Time required:

  1. 3-4 hours on Day 1
  2. 2-4 hours on Day 2

Special materials:

Procedure:

Day 1

Isolating the fragment:

  1. Run the restriction digest and the appropriate size markers on a 1X Tris-Borate agarose gel with ethidium bromide. Be sure to leave at least 1-2 wells between samples. Run the gel until the DNA bands are well separated (visualize on the long- wavelength UV lightbox.
  2. Cut a slit just ahead of the band of interest using a sharp sterile razor blade or scalpel. Using blunt-edged forceps (such as Millipore forceps) carefully insert an NA-45 paper into the slit (prewet and cut NA-45 to the width of the band, see preparation of the NA-45 below).
  3. Place the gel in fresh 1X Tris-Borate buffer, and run the gel until the fragment has moved out of the gel and stopped by the NA-45. Monitor the progress of the band with the hand-held long wavelength UV light. Do not allow other bands of higher molecular weight to run onto the NA-45.
  4. Remove the NA-45 paper, rinse in NET buffer and place in a labeled eppendorf tube. Add sufficient high-salt NET buffer to cover most of the membrane (typically 150-300 Ál). Spin 5 seconds in a microcentrifuge to submerge the entire strip. Place at 65 degrees C for 1 hour, mixing frequently, and respinning if the membrane rides up the side of the tube.
  5. Transfer the buffer (+ DNA fragment) to a clean labeled tube. Wash the membrane (in the original tube) with 50 Ál high salt NET buffer and add the wash to the DNA fragment tube.

Cleaning the DNA:

  1. To remove ethidium bromide, extract twice with 3 volumes water-saturated n-butanol.
  2. Precipitate the DNA with 2.5 volumes of ethanol at -20 degrees C for at least 1 hour (can sit overnight in the freezer).

Day 2

  1. Pellet the DNA (for 20 minutes at high speed in a microcentrifuge) and resuspend in 50 Ál TE. Reprecipitate with sodium acetate to remove any residual NaCl: Add 5 Ál 3M Na-acetate, and 120 Ál ethanol, hold at -20 degrees C for 2 hours or more, pellet as before and resuspend in an appropriate amount of TE.

Solutions:

References:

Sambrook, J., Fritsch, E.F., and T. Maniatis.(1989) Molecular Cloning, A Laboratory Manual. Second edition. Cold Spring Harbor Laboratory Press. pp 6.24-6.27.