Electrocompetent E. coli 1.

[Luke]

Competent E.coli Cells for electroporation

1.   Autoclave 2x 500mL LB (each in 2L flask) and 50mL LB (in a 250mL flask), 1L distilled water and 2x 250mL centrifuge bottles
2.   Innoculate the 50mL starter with a stab of commercial electrocompetent E.coli (this protocol has been used successfully on DH10B), grow overnight
3.   Innoculate each 500mL LB with 25mL of overnight culture
4.   Measure absorbance at 600nm (blanked to LB) and grow to 0.8-1.0
5.   Put flasks on ice
6.   Centrifuge at 5000 RPM for 10 minutes to pellet all bacteria
7.   Resuspend and wash in cold sterile ddH20, bring volume up to fill both bottles
8.   Centrifuge at 5000 RPM for 10 minutes
9.   Resuspend and wash in cold sterile ddH20, bring volume up to 200mL in one bottle
10.   Centrifuge at 5000 RPM for 10 minutes
11.   Resuspend in 10mL sterile ddH20 and transfer to 15mL conical tube
12.   Centrifuge at 3000 RPM, 10 minutes, aspirate supernatant
13.   Resuspend in 10mL cold electroporation buffer (10% glycerol, 20% Polyethylene glycol)
14.   Centrifuge at 3000 RPM, 20 minutes, gently aspirate clear layer being careful not to aspirate slightly loose layer of cells
15.   Resuspend in 5mL cold electroporation buffer
16.   Centrifuge at 3000 RPM, 20 minutes, gently aspirate and avoid aspirating loose cell layer
17.   Resuspend loose with tight layer of cells and aliquot into 100uL aliquots on dry ice.
18.   Store at -80C