MIT7_13f08_lab15_Protocol_Pseudomonas

MIT7_13f08_lab15_Protocol_Pseudomonas - -slowly mix cell...

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MIT OpenCourseWare http://ocw.mit.edu 7.13 Experimental Microbial Genetics Fall 2008 For information about citing these materials or our Terms of Use, visit: http://ocw.mit.edu/terms .
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7.13 Fall 2008 Page | 1 PSEUDOMONAS ELECTROPORATIONS Materials: - o/n culture of recipient strain - plasmid for electroporation - 300mM sucrose (filter sterilized) - LB - 2 mm gap width cuvette Protocol: - grow overnight culture of Pseudomonas strain in LB at 37°C (1ml/electroporation) - transfer 1ml o/n culture into a microcentrifuge tube and spin for 1 minute at 13,000 rpm - carefully aspirate off supernatant and resuspend in 1ml 300 mM sucrose - spin again at 13,000 rpm for 1 minute and repeat sucrose wash - final spin at 13,000 rpm for 2 minutes, aspirate remaining liquid and resuspend in 100 ul sucrose - if working with a replicative plasmid, add ~10 - 50 ng plasmid to 100 ul cell suspension. If working with a non-replicative plasmid (i.e. suicide plasmid) add ~ 300 – 500 ng plasmid.
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Unformatted text preview: -slowly mix cell suspension several times by pipetting and transfer into a 2 mm gap width cuvette -gently tap and/or flick cuvette several times until cell suspension settles at the bottom between the two metal plates -insert cuvette into electroporation apparatus and slide cuvette holder all the way back until cuvette is in contact with the electrodes -electroporation settings should be 2.5 kV, 25 uF, 200 -press and hold two red buttons simultaneously until tone sounds o time constant (displayed after electroporation) should be 4-5 msec -immediately resuspend cells in 1 ml room temperature LB and grow at 37C in rotator for 1-1.5 hours -transfer cells to a microcentrifuge tube, spin at 13,000rpm for 2 minutes and aspirate supernatant leaving the cell pellet in ~50 ul LB -resuspend cells in remaining 50 ul LB and plate on LB + antibiotic plate, incubate plates overnight at 37C o antibiotic should correspond to resistance conferred by plasmid...
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MIT7_13f08_lab15_Protocol_Pseudomonas - -slowly mix cell...

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