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.: Electro Competent Cells :.

A -80 degree C frozen stock is prepared from Gibco-BRL DH10B competent cells. From this frozen stock, a freshly streaked agar plate is used to prepare the competent cells. This protocol was obtained from the Parkhurst lab.

SOB Agar
100 ml SOB-Mg
1 ml 2M MgCl2-MgSO4
1.5 gm agar
Mix in 250 ml flask, autoclave. Pour 4 plates.
*Used for streaking commercial DH10B cells

SOB Medium
100 ml SOB-Mg
1 ml 2M MgCl2-MgSO4
Mix in 100 ml bottle, autoclave.
*Used for preparation of frozen stock.

SOB-Mg Medium
30 gm Bacto-tryptone
7.5 gm Bacto yeast extract
0.87 gm NaCl
0.285 gm KCl
dH2O to 1.5 liters
Distribute to the following containers and autoclave:
50 ml into 500 ml flask
500 ml into two-liter flask (x 2 flasks)
*Used for preparation of competent cells

60% SOB – 40% Glycerol
60 ml SOB-Mg
0.6 ml 2M MgCl2-MgSO4
40 ml glycerol
Mix in 200 ml bottle, autoclave.
*Used for preparation of -80 degree C frozen stock

SOB-Mg Agar
100 ml SOB-Mg
1.5 gm agar
Mix in 250 ml flask, autoclave. Pour 4 plates.
*Used in streaking the -80 degree C frozen stock

10% glycerol
40 ml glycerol
360 ml H2O
Mix in 500 ml bottles (x 4 bottles), autoclave, chill O/N at 4 degree C.

Preparation of -80oC Frozen Stock

1. Streak "MAX EFFICIENCY DH10B Competent Cells" (GIBCO-BRL) on SOB plate.
* No drug selection
2. Incubate O/N until colonies are about 2-3 mm in diameter, approx. 24 hours.
3. Inoculate a Falcon 2059 tube containing 2 ml SOB medium with a single colony.
4. Incubate at 37 degree C while shaking until it gets slightly cloudy, approx. 2 hours.
* The culture should be at about 1-2 x 108 cells/ml
5. Mix 0.5 ml culture with 0.5 ml 60% SOB-40% glycerol in chilled 1.5 ml tubes. Flash-freeze in dry ice/EtOH bath. Store at -80 degree C.
Preparation of Competent Cells

1. Streak DH10B cells from frozen stock on a SOB-Mg plate. Incubate O/N at 37 degree C.
2. Disperse a single 2-3 mm colony into 1 ml SOB-Mg by vortexing. Inoculate into a 500 ml flask containing 50 ml SOB-Mg. Incubate O/N at 37 degree C while shaking at 275 rpm (preferably <12 hours).
* No drug selection
3. Inoculate 500 ml of SOB-Mg in a two-liter flask (x 2) with 5 ml culture. Incubate at 37 degree C while shaking until A550 = 0.75, approx. 2 hours 50 minutes.
4. Transfer each flask of cells into two chilled 250 ml centrifuge bottles. Pellet cells at 2600 g (4 krpm in SORVALL GSA rotor) at 4 degrees C for 15 min. Discard supernatant and remove residual liquid by pipette.
5. Resuspend the cells in an equal volume of chilled 10% glycerol by repeated pipetting while keeping the cells on ice. Pellet cells at 2600 g at 4 degree C for 15 min. Immediately decant the supernatant.
6. Repeat step 5.
7. Resuspend the cells in 100 ul or less/bottle of 10% glycerol.
8. Combine all cells together. Expect about 2 ml total volume.
9. Dilute a 10 ul aliquot to 3.0 ml with 10% glycerol. Measure the O.D. at 550 nm.
Example: O.D. (550 nm) of 1/300 dilution = 0.75
O.D. (550 nm) of original suspension = 225
The O.D. at 550 nm of the original suspension should be 200-250 units/ml, if not then dilute it with
chilled 10% glycerol.
10. Aliquot the cells to chilled 1.5 ml tubes, 120 ul/tube. Freeze in a dry ice/EtOH bath. Store at -80 degrees C.
Use 40 ul cells per transformation.