Multiporator / Electroporator 2510
Protocol No. 4308 915.506 12/2001
Borrelia burgdorferi B31
Bacteria, gram negative
Linear DNA (in water)
Barbour-Stoenner-Kelly (BSK) II medium without gelatin
PBS, without divalent cations; EPS (272 mM sucrose, 15% glycerol)
EPS (272 mM sucrose, 15% glycerol)
BSK II medium
2 mm gap width
Samuels D.S., et al 1994 Journal of Bacteriology 176
Making electrocompetent cells:
- Grow cells in 500 ml BSK II at 32 C to a cell density of 3-7
x 107 cells/ml.
- Harvest by centrifugation (4,000 x g, 20 min., 4 C). Wash twice
in 60 ml cold PBS and pellet by centrifugation (3,000 x g, 10 min.,
- Wash three times in 20 ml cold EPS and pellet by centrifugation (2,000
x g, 10 min., 4 C).
- Resuspend the cells in 0.6 ml cold EPS.
Electroporation of c
- Add 0.3 to 1 g DNA to 50 l of electrocompetent cells
on ice. Homogenize by gently mixing with pipette several times. Transfer
mixture into a prechilled cuvette.
- Wipe moisture from the cuvette and insert the cuvette into the device.
Time constant (T)
- Immediately add 1 ml BSK II medium, transfer cells to a 15 ml tube
and add another 9 ml of BSK II. Incubate for 24-48 hours at 32 C
- Plate 0.1 ml of culture on solid BSK II medium. Pellet the remaining
9.9 ml (4,000 x g, 10 min.), resuspend in 1 ml of supernatant and plate
on selective plates. Incubate for 14 days, at 32 C in a humidified
5% CO2 chamber.
Transformation efficiency up to 103 transformants/g
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. Borrelia burgdorferi