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+ | <h>Preparation of Bacillus subtilis competent cells</h> | ||
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+ | <ol>1.Streak out the strain to be made competent on an LB agar plate as a large patch and incubate overnight at 30°C</ol> | ||
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+ | <ol>2. The following morning scrape the cell growth off the plate and use to inoculate fresh, pre-warmed, SpC medium (20 ml) to give an OD600 reading of about 0.5. </ol> | ||
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+ | <ol>3. Incubate the culture at 37°C with vigorous aeration and take periodic OD readings (OD600) to assess cell growth.</ol> | ||
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+ | <ol>4. When the rate of cell growth is seen to depart from exponential (i.e. no significant change in cell density over 20-30 min) inoculate 200 ml of pre-warmed, SpII medium with 2 ml of stationary-phase culture and continue incubation at 37°C with slower aeration </ol> | ||
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+ | <ol>5. After 90 min incubation, pellet the cells by centrifugation (8,000 g, 5min) at room temperature.</ol> | ||
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+ | <ol>6. Carefully decant the supernatant into a sterile container and save.</ol> | ||
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+ | <ol>7. Gently resuspended the cell pellet in 18 ml of the saved supernatant and add 2 ml of sterile glycerol; mix gently </ol> | ||
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+ | <ol>8. Aliquot the competent cell (0.5 ml) in sterile tubes, freeze rapidly in liquid nitrogen or a dry-iced/ethanol bath or ice/isopropanol bath and store -70.</ol> | ||
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<p>Competency Step 1 :</p> | <p>Competency Step 1 :</p> | ||
<img src="aaa" width="500px"/> | <img src="aaa" width="500px"/> |
Revision as of 14:15, 29 July 2016