Team:Wisconsin-Madison/Preparation of Competent Cells

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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.

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