Multiporator / Electroporator 2510
Protocol No. 4308 915.540 07/2002
Bacteria, gram positive
Plasmid DNA (pGK12 and pPN-1)
MRS-G with 0.5 M sorbitol, 3 % glycine and 40 mM DL-threonine
0.5 M sorbitol, 10 % glycerol
0.5 M sorbitol, 1 mM K2
, 1mM MgCl2
, pH 7.0
MRS with 0.5 M sorbitol, 20 mM MgCl2
and 2 mM CaCl2
1 mm gap width
Caldwell, S. L. et al 1996
Applied and Environmental Microbiology 62, No. 3 936-941
Making electrocompetent cells:
Cultivate cells by adding 12 ml overnight preculture
(grown in MRS-G with 0.5 M sorbitol) to 800 ml growth medium. Incubate
for 2 to 4 hours at 37 C to a cell density of O.D.600
Harvest by centrifugation.
Wash twice in 25 ml washing solution.
end in 1 ml electroporation solution.
Electroporation of cells:
- Add 400-600 ng plasmid DNA to 80 l of electrocompetent cells.
Homogenize by gently mixing with pipette several
times. Transfer mixture into cuvette.
- Wipe moisture from the cuvette and insert the cuvette into the device.
Time constant (T)
- Immediately add 2 ml outgrowth medium and keep on ice for approx.
5 min. Incubate for 2 h at 37 C.
- Plate aliquots onto selective agar plates; incubate 2-5 days at 37
Transformation efficiency up to 4.6 x 103
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. Pediococcus spp.