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Team:StanfordBrownSpelman/Lab Techniques10
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<b>Phase 1</b> | <b>Phase 1</b> | ||
| - | <br />1. | + | <br />1. Inoculate 25% (5mL) of desired final volume of LB with BS168 in the morning, |
in container >200% final volume (50mL falcon tube) | in container >200% final volume (50mL falcon tube) | ||
| - | <br />2. | + | <br />2. Incubate @37C for ~6hrs, then top up to final volume (20mL) LB to incubate |
overnight | overnight | ||
| - | <br />3. | + | <br />3. Spin and pellet @3000g for 5 min |
| - | <br />4. | + | <br />4. Wash with cold (4C) sterile deionized water by resuspending (in 25-50% |
original volume) and spinning @3000g for 5min | original volume) and spinning @3000g for 5min | ||
| - | <br />5. | + | <br />5. Discard supernatant, repeat twice more |
| - | <br />6. | + | <br />6. Finally resuspend in 1% of the original culture volume (from which the pellet |
was formed) with cold (4C) 30% PEG solution | was formed) with cold (4C) 30% PEG solution | ||
| - | <br />7. | + | <br />7. Aliquot into 100μl samples (use pre-chilled tubes) |
| - | <br />8. | + | <br />8. Freeze immediately @ -80C |
| - | <br />9. | + | <br />9. After waiting overnight, proceed to phase 2 |
<br /><br /><b>Phase 2 (electroporation)</b> | <br /><br /><b>Phase 2 (electroporation)</b> | ||
| - | <br />1. | + | <br />1. Thaw cells @ 4C until liquid |
| - | <br />2. | + | <br />2. Rransfer to cold .2cm electroporation cuvette |
| - | <br />3. | + | <br />3. Apply current with cuvette uncapped, @ 25μF, 2.5kV (12.5kV/cm), 400ohms |
| - | <br />4. | + | <br />4. Immediately add 2ml of prewarmed SOC to cuvette, cap, and mix by inverting |
several times | several times | ||
| - | <br />5. | + | <br />5. Transfer cuvette contents to 15ml falcon tube by pipetting or decanting and |
incubate for 90 min @37C | incubate for 90 min @37C | ||
| - | <br />6. | + | <br />6. Plate on preheated selective agar (if unsure about efficiency, try 100μl, 15μl) |
<br /><br /><i>Escherichia coli</i> (adapted from Dr. Shih’s protocol) | <br /><br /><i>Escherichia coli</i> (adapted from Dr. Shih’s protocol) | ||
Doubling time for E.coli in ideal conditions, 37ºC = 20 minutes. | Doubling time for E.coli in ideal conditions, 37ºC = 20 minutes. | ||
Latest revision as of 04:35, 16 October 2014
