Method: Preparation and Restriction Digestion of Human Cells in Agarose Beads

May 1, 1990

Jim Howe

Purpose:

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.

Time required:

2-3 days

Special Equipment:

CHEF DR unit

Special Reagants:

1. Low gelling agarose
2. Phenylmethylsulfonylfluoride (PMSF)

caution: PMSF is extremely toxic to mucous membranes, do not inhale or allow contact with skin

Procedure:

Day 1

1. Obtain lymphoblastoid cell line with a cell count of 1 x 108 cells. Wash and resuspend cells in 5 ml phosphate buffered saline (PBS).
2. 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.
3. 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.
4. 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.
5. 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.
6. 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.
7. Resuspend in 4 volumes (20 ml) of proteinase K solution and incubate at 50 degrees C overnight.

Day 2

1. Wash beads several times in TE7.5 solution containing 0.1 mM PMSF, and leave at room temperature for 30 minutes during each wash.
2. 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:

1. Wash beads several times in 10 mM Tris-HCl (pH 7.4) to completely remove EDTA.
2. 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).

Solutions:

• PBS:

  1.5 mM KH2PO4 8 mM Na2HPO4 3 mM KCL 0.14 M NaCl adjust to pH 6.8

• Lysis solution:

  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

• TE7.5 solution:

  10 mM Tris HCl 1 mM EDTA adjust pH to 7.5

References:

Imai, T., and Olson, M.V. (1990) "Second generation approach to the construction of yeast artificial chromosome libraries." Genomics 8: 297-303.