Team:Chiba/Notebook/Protocol

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Zymo comp.
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Day 1 morning<br>
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100 ml SOB medium in 1L or 500 mL flask and sterilize<br>
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E.coli culture grown in 2 mL of fresh LB medium.<br>
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Day 1 night<br>
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Inoculate preculture (100 μL-1 mL) to sterile SOB medium.Shake culture vigoruoussly at 20-25 ℃ until OD is 0.4-0.6.<br>
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Day 2<br>
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Transfer the culture to ice 10min.<br>
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Prepare Wash buffer and Competent buffer by adding 3 mL Dilution buffer to 3 mL of Wash buffer(x2) and to 2.5 mL of Competent buffer(2x), respectively.(on ice)<br>
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Pellet the cells by centrifugarion at 2500rpm for 6 min.<br>
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Remove supernatant and genlly resupend the cells in 6 mL ice-cold Wash buffer(1x).<br>
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Pellet the cells by centrifugarion at 2500rpm for 6 min.<br>
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Completely remove the supernatant and gently resuspend the cells in 6 mL ice-cold Competent buffer(1x).<br>
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Aliquot (on ice) 100μL of cell suspension into sterile 1.5 mL microtube and store in deep freezer.<br>
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Latest revision as of 04:03, 16 September 2013

iGEM-2013 Chiba

iGEM-2013 Chiba

Zymo comp.

Day 1 morning
100 ml SOB medium in 1L or 500 mL flask and sterilize
E.coli culture grown in 2 mL of fresh LB medium.
Day 1 night
Inoculate preculture (100 μL-1 mL) to sterile SOB medium.Shake culture vigoruoussly at 20-25 ℃ until OD is 0.4-0.6.
Day 2
Transfer the culture to ice 10min.
Prepare Wash buffer and Competent buffer by adding 3 mL Dilution buffer to 3 mL of Wash buffer(x2) and to 2.5 mL of Competent buffer(2x), respectively.(on ice)
Pellet the cells by centrifugarion at 2500rpm for 6 min.
Remove supernatant and genlly resupend the cells in 6 mL ice-cold Wash buffer(1x).
Pellet the cells by centrifugarion at 2500rpm for 6 min.
Completely remove the supernatant and gently resuspend the cells in 6 mL ice-cold Competent buffer(1x).
Aliquot (on ice) 100μL of cell suspension into sterile 1.5 mL microtube and store in deep freezer.

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