Preparation of High-Molecular-Weight Human DNA Restriction Fragments in Agarose Plugs

Purpose
To prepare intact mammalian chromosomal DNA for use in pulsed-field electrophoresis mapping experiments, using rare cutting restriction enzymes.

Time Required
4 days.

Materials
Phenylmethylsulfonyl fluoride (PMSF)

Procedure
Day 1–2

1. Place freshly grown mammalian cells in a 15-µL Falcon tube and centrifuge in a Beckman tabletop centrifuge at 1000 rpm for 15 minutes. Pour off supernatant, and resuspend pellet in 10 mL of phosphate buffered saline (PBS). Centrifuge at 1000 rpm for 15 minutes again, then resuspend in PBS to achieve a cell concentration of 1–2 x 107 cells/mL (refer to methods in tissue culture regarding hemocytometer for cell counting procedure).
2. Mix an equal volume of cells (1–2 µL) with premelted 1% low gelling agarose which has been cooled to 45°;C–50°;C, using a Pasteur pipette; immediately distribute the mixture to plug molds for the CHEF apparatus. Place the mold on ice for 15 minutes.
3. Remove plugs, then incubate in 6 well tissue culture plates (5 µL/well, several plugs/well) in ESP solution at 50°;C with gentle shaking for 2 days; be sure to seal the 6 well plate securely with parafilm to prevent evaporation of the samples. Plugs can now be stored in ESP at 4°;C, or one can proceed as follows.

Day 3

1. For each digest condition, use approximately ¼ of a plug, roughly the size that would fill a well on the CHEF DR apparatus. Place each in 1 mL of 1-mM PMSF (caution: extremely toxic to mucous membranes, do not inhale or allow contact with skin) diluted from 0.1 M stock in TE (pH 8), and slowly rotate plates at room temperature for 2 hours. Replace with new PMSF aliquot and repeat. Remove PMSF, and wash in 1 mL of TE (pH 8) 3 times for 1–2 hours each by slow rotation at room temperature.
2. Place each plug in 250µL of appropriate restriction buffer (1X) in eppendorf tubes at room temperature for 30 minutes. Aspirate buffer carefully, then replace with fresh buffer and hold at room temperature for 30 minutes. Replace with 250µL of fresh buffer, then add 25 µg of BSA to each tube. Add 20 units of enzyme per mg of DNA to tubes containing the appropriate buffer (¼ of one plug should be approximately equal to 5–10 µg of DNA if the cell counts are correct). Incubate overnight at the appropriate temperature; additional enzyme can be added the next morning and incubation carried out for 4 more hours if so desired.

Day 4

1. Replace buffer with 1 mL of ES solution, and incubate at 50°;C for 2 hours with gentle shaking.
2. Replace ES with 250 µL ESP solution and incubate at 50°;C for an additional 2 hours.
3. Load the plugs into a gel poured for the CHEF DR apparatus. Run under conditions appropriate for the fragment sizes expected. Fragments in the 200–2200 kb size range can be effectively separated on a 1.0% agarose gel run at 200 V with a switch time of 60 seconds for 15 hours, and 90 seconds for 9 hours.

Solutions

• ESP solution:
  0.5 M EDTA, pH 9.0–9.5
  1% sodium laurylsarcosine
  1 mg/mL Proteinase K
• PMSF stock:
  0.1 M PMSF in 2-Propanol
• ES solution:
  0.5 M EDTA, pH 9.0–9.5
  1% sodium laurylsarcosine