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Method: Preparation and Restriction Digestion of Human Cells in Agarose Beads
May 1, 1990
To prepare high molecular weight DNA in a medium with high surface area, to facilitate complete digestion with restriction enzymes.The DNA prepared in agarose beads is also suitable for use in constructing YAC libraries.
- Low gelling agarose
- Phenylmethylsulfonylfluoride (PMSF)
caution: PMSF is extremely toxic to mucous membranes, do not inhale or allow contact with skin
- Obtain lymphoblastoid cell line with a cell count of 1 x 108 cells. Wash and resuspend cells in 5 ml phosphate buffered saline (PBS).
- Premelt 5 ml of 1% low gelling agarose (in PBS) in a 300 ml Erhlenmeyer flask, and cool to 45 degrees C. Prewarm the cells for less than a minute at 45 degrees C, then mix with the agarose.
- Add 20 ml paraffin oil (prewarmed to 45 degrees C) to the flask, and swirl vigorously. Pour the resulting emulsion into a 500 ml flask containing 100 ml of ice cold PBS, which should be in an ice bath and stirring with a magnetic stir bar. Allow to stir for several minutes.
- Divide this volume into six 50 ml conical tubes, and pellet the beads by centrifugation at 2000 rpm for 10 minutes, then aspirate off the supernatent using a pasteur pipette.
- Pool the beads into one 50 ml conical tube, then fill tube with PBS (beads can be stored overnight like this). Centrifuge at 2000 rpm for 2 minutes, then aspirate fluid.
- Add 4 volumes of lysis solution (20 ml), then incubate at room temperature for 30 minutes. Centrifuge at 2000 rpm for 10 minutes, then aspirate fluid.
- Resuspend in 4 volumes (20 ml) of proteinase K solution and incubate at 50 degrees C overnight.
- Wash beads several times in TE7.5 solution containing 0.1 mM PMSF, and leave at room temperature for 30 minutes during each wash.
- Wash three times in TE7.5 solution; beads can now be stored at 4 degrees C in TE7.5. The expected yield from 108 cells is 5-7 ml of beads (approx. 50 µg/ml), which should be stored in a total volume of 10 ml.
Restriction Enzyme Digests:
- Wash beads several times in 10 mM Tris-HCl (pH 7.4) to completely remove EDTA.
- Equilibrate 50 µl of beads with 6 µl 10X restriction buffer in an eppendorf tube for 5 minutes; repeat with a change of buffer 2-3 times. Add appropriate amount of enzyme for the application (partial vs. complete digestion; this volume of beads contains approximately 2 µg of DNA).
1% sodium dodecyl sulfate 25 mM EDTA pH 8.0 10 mM Tris HCl pH 8.0
Proteinase K solution:
1% Sarkosyl 25 mM M EDTA pH 8.0 50 µg/ml proteinase K
10 mM Tris HCl 1 mM EDTA adjust pH to 7.5
Imai, T., and Olson, M.V. (1990) "Second generation approach to the construction of yeast artificial chromosome libraries." Genomics 8: 297-303.