Multiporator / Electroporator 2510
Protocol No. 4308 915.527 04/2002
Bacteria, gram positive
Ice-cold 0.5 M sucrose
Ice-cold 0.5 M sucrose buffered with 1 mM potassium
acetate (pH 5.5)
SLB medium with 0.5 M sucrose
1 mm gap width
Jore, J. P M. et al 2001 Applied
and Environmental Microbiology 67, No. 2 499-503
Making electrocompetent cells:
Cultivate cells anaerobically at 30 C in SLB medium
to the stationary growth phase. Dilute 1:50 in fresh SLB medium and
incubate again for about 20 h.
Harvest in the exponential growth phase by centrifugation
and wash extensively with ice-cold 0.5 M sucrose.
Wash once in ice-cold electroporation solution.
Resuspend in ice-cold electroporation solution (abo
1/100 of the original culture volume).
Electroporation of cells:
- Add plasmid DNA to 80-100 l of electrocompetent cells. Homogenize
by gently mixing with pipette several times.
Transfer mixture into prechilled cuvette.
- Wipe moisture from the cuvette and insert the cuvette into the device.
Time constant (T)
- Immediately add 900 l cold outgrowth medium and incubate for
2.5-3 h at 30 C.
- Plate cells onto selective SLB plates; incubate 5-7 days at 30 C
under anaerobic conditions.
Transformation efficiency up to 1 x 108
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