Centrifuge down cells.
Wash once in SET.
Resuspend pellets in SET at conc. of 4 x 108 cells/ml.
Add an equal volume of low melting agarose in 0.125 M EDTA to give 0.7% final concentration.
NOTE: Cells should be close to 37 C and agarose no more than 40 C.
Pour the solution into a mold.
Refrigerate for 20’ before removing blocks from mold.
Cut blocks into small cubes.
Soak for 30 min in 0.5 M EDTA at room temperature.
Remove solution and add 3 ml (enough solution to cover cubes) of 2 mg/ml EDTA-sarkosyl-pronase solution..
Put at 50 C overnight.
Remove the EDTA-Sarkosyl-Pronase solution and add fresh solution.
Incubate for 24 hours.
Remove solution and rinse cubes in SET.
Store in SET at 4 C.
SOLUTIONS:
SET:
0.15M NaCl
0.1M EDTA ph 8.0
10 mM Tris ph 8.0
PRONASE:
10 mg/ml stock in SET
Put solution at 37 C for 30 minutes.
Quick cool and store frozen
GEL:
1.5% agarose in 0.5X TBE
Make a single slot.
Run at 100 mA (=280 Volts)
60 sec. pulse time.
10 C.
To resolve high molecular weight chromosomes, use 0.8 or 1% agarose in 0.5 TBE.
60 sec. pulse for 16 hours.
120 sec. or 140 sec. pulse for 35 hours.
150 Volts.