Team:Wisconsin-Madison/Preparation of Competent Cells

Transformation of Plasmids into Competent Cells

The process consists of growing cells to mid-log stage, harvesting, and performing multiple washes with sterile 10% glycerol to remove salts which interfere with electroporation.

General Considerations:

• Keep everything cold, on ice
• Glycerol pellets are not firm; try to remove as much supernate as possible, but be careful not to lose the pellet
• All containers that come in contact with cells should be sterile
• Keep centrifuge bottles dedicated for making Electrocompetent cells
• Have 1 liter of 10% sterile glycerol chilled on ice, to less than 4C... or in a cold box overnight.
• Keep manipulation of cells to a minimum, be gentle.
• Resuspend pelleted cells using a sterile plastic pipette. Work quickly.
• Harvest cells at 0.6 – 0.75 O.D. (A600nm)

A) Fermentation (Inoculum)

• Streak for single colony from -70C glycerol stock
• Start 50 ml, No Salt LB inoculum, 37C, overnight
• Fermentation
• Use 25 ml of the above Inoculum per liter of No Salt LB media (prewarm media to 37C)
• Grow at 37C, shake at approximately 200 rpm
• Grow to 0.6 – 0.75 O.D. (A600nm)......transfer to ice immediately to chill

B) Processing

1) Spin the chilled culture at 8,000 rpm, 10 minutes, 2 degrees C (use four 250 ml centrifuge bottles). Remove the supernate carefully. Save the pellets.

2) Resuspend all four pellets in a total volume of 200 ml cold 10% glycerol. Combine all resuspended pellets in one 250 ml centrifuge bottle.

3) Spin at 8,000 rpm, 10 minutes. Remove the supernate carefully.

4) Resuspend pellet in 150 ml cold 10% glycerol.

5) Spin at 8,000 rpm, 10 minutes. Remove the supernate carefully.

6) Resuspend pellet in 100 ml cold 10% glycerol.

7) Spin at 8,000 rpm, 10 minutes. Remove the supernate carefully.

8) To the pellet, add 2 ml 10% glycerol. Resuspend carefully with a 1 ml Pipetteman.

9) Transfer 110 ul of resuspended cells into cold***(-70C) 1.5 ml microcentrifuge tubes.

10) Transfer immediately to a -70C freezer (Do not use liquid nitrogen).

11) Freeze overnight before using cells.

• The microcentrifuge tubes should be in a plastic tray, having been stored overnight at -70C freezer. Remove the tray and tubes from the -70C freezer immediately prior to aliquoting cells into the microcentrifuge tubes.