AX2 cells that have been grown in log phase for 2 days are harvested at a density of
4-6 x 10E6 cells/ml
Harvest 4 x 10E8 cells. Wash 1x in ice cold E-buffer. Resuspend in 10 ml ice cold E-buffer
(10 mM Na/K phosphate buffer pH 6.1, 50 mM sucrose). Keep on ice.
Add 100 mg plasmid (pBSR1 or pBSR3 x
BamHI)
Take 4 control samples (no enzyme); 0.4 ml each.
Add 50 units (5m l) DpnII to remaining cell
suspension (5
units DpnII/ml).
Electroporate at 1.2 kV and 3 m F, using BioRad Gene Pulser
with 5 Ohm resistor in series with chamber. Time constant must be 0.5-0.7. Use 0.2 cm
cuvettes (Green label).
After electroporation incubate10 minutes on ice.
Add 2 m l of "healing solution" (100 mM CaCl2,
100 mM MgCl2) and transfer to petri dish. Let sit 15 minutes.
Add 12 ml HL5 medium and spread the cells evenly in petri dish.
Next day add 5 mg/ml (6 m l; 10
mg/ml) Blasticidin.
Transformants appear in appr. 5 days.
Screen about 3 times the number of
transformants.