1) Grow cells in the appropriate medium to OD600 of 0.5 (approx. 10^7 cells/ml).
3) Wash cell pellet with 25 ml of sterile H2O.
4) Resuspend in 1 ml of TE/LiAc and transfer to eppendorf tubes.
5) Spin down at 5,000 x rpm for 5 min in tabletop microcentrifuge.
6) Remove sup and add enough TE/LiAc to pellet to achieve final concentration of 109 cells/ml, taking into account of the volume of the cells too.
7) Incubate at RT for 1 hr.
8) At approx. 15 min before the end of the incubation, prepare the carrier Herring Sperm DNA. Remove the desired amount of DNA from stock and transfer to an eppendorf tube. Boil in the heating block for 10 min. Immediately cool on ice. Pulse-spin to return condensation to the bottom of the tube before use.
9) Remove 50 ml of cells for each transformation and place in a new eppendorf tube.
10) Spin down at top speed for 10 sec.
11) Remove sup and add the following components (in the exact order as follows):
240 ml 50% PEG
10 ml boiled and chilled Herring Sperm DNA (10 mg/ml)
20 ml unpurified PCR product (if doing gene knock-outs) or 1 mg of plasmid DNA (if doing plasmid transformation)
54 ml H2O
12) Mix by passing the tubes over a spiky finger rack.
13) Incubate at 42 oC for 40 min.
14) Spin down at 5,000 x rpm for 5 min.
15) Remove sup and resuspend in 600 ml of agd H2O.
16) Plate 150 ml on each plate.
17) Incubate at the appropriate temperature.
50% Polyethylene glycol MW4000
1 M Tris-HCl pH 8.0
1 M Lithium Acetate pH 7.5
0.2 M EDTA pH 8.0
10 mM Tris-HCl, pH 8.0
1 mM EDTA, pH 8.0
100 mM LiAc
To make 50 ml TE/LiAc, mix:
0.5 ml 1M Tris-HCl, pH 8.0
0.25 ml 0.2 M EDTA, pH 8.0
5 ml 1M LiAc
44.25 ml agd H2O
44.25 ml agd H2O