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Team:StanfordBrownSpelman/Lab Techniques10
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Revision as of 04:33, 16 October 2014
Bacillus subtilis Transformation
Phase 1
1.inoculate 25% (5mL) of desired final volume of LB with BS168 in the morning,
in container >200% final volume (50mL falcon tube)
2.incubate @37C for ~6hrs, then top up to final volume (20mL) LB to incubate
overnight
3.spin and pellet @3000g for 5 min
4.wash with cold (4C) sterile deionized water by resuspending (in 25-50%
original volume) and spinning @3000g for 5min
5.discard supernatant, repeat twice more
6.finally resuspend in 1% of the original culture volume (from which the pellet
was formed) with cold (4C) 30% PEG solution
7.aliquot into 100μl samples (use pre-chilled tubes)
8.freeze immediately @ -80C
9.after waiting overnight, proceed to phase 2
Phase 2 (electroporation)
1.thaw cells @ 4C until liquid
2.transfer to cold .2cm electroporation cuvette
3.apply current with cuvette uncapped, @ 25μF, 2.5kV (12.5kV/cm), 400ohms
4.immediately add 2ml of prewarmed SOC to cuvette, cap, and mix by inverting
several times
5.transfer cuvette contents to 15ml falcon tube by pipetting or decanting and
incubate for 90 min @37C
6.plate on preheated selective agar (if unsure about efficiency, try 100μl, 15μl)
Escherichia coli (adapted from Dr. Shih’s protocol)
Doubling time for E.coli in ideal conditions, 37ºC = 20 minutes.
1.inoculate 25% (5mL) of desired final volume of LB with BS168 in the morning, in container >200% final volume (50mL falcon tube)
2.incubate @37C for ~6hrs, then top up to final volume (20mL) LB to incubate overnight
3.spin and pellet @3000g for 5 min
4.wash with cold (4C) sterile deionized water by resuspending (in 25-50% original volume) and spinning @3000g for 5min
5.discard supernatant, repeat twice more
6.finally resuspend in 1% of the original culture volume (from which the pellet was formed) with cold (4C) 30% PEG solution
7.aliquot into 100μl samples (use pre-chilled tubes)
8.freeze immediately @ -80C
9.after waiting overnight, proceed to phase 2
Phase 2 (electroporation)
1.thaw cells @ 4C until liquid
2.transfer to cold .2cm electroporation cuvette
3.apply current with cuvette uncapped, @ 25μF, 2.5kV (12.5kV/cm), 400ohms
4.immediately add 2ml of prewarmed SOC to cuvette, cap, and mix by inverting several times
5.transfer cuvette contents to 15ml falcon tube by pipetting or decanting and incubate for 90 min @37C
6.plate on preheated selective agar (if unsure about efficiency, try 100μl, 15μl)
Escherichia coli (adapted from Dr. Shih’s protocol) Doubling time for E.coli in ideal conditions, 37ºC = 20 minutes.
