(→Heat shock competent cells tranformation) |
(→Protocols) |
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==Protocols== | ==Protocols== | ||
− | ===Heat shock competent cells | + | ===Heat shock competent cells=== |
+ | ====Preparation==== | ||
'''Day 1''' Inoculate cells in 3.5mL LB medium. | '''Day 1''' Inoculate cells in 3.5mL LB medium. | ||
Incubate at 37°C overnight. | Incubate at 37°C overnight. | ||
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Dissolve HEPES, CaCl<sub>2</sub> and KCl in water. Adjust pH to 6.7 with KOH. Add MnCl<sub>2</sub>. Filter to sterilize and keep at 4°C. | Dissolve HEPES, CaCl<sub>2</sub> and KCl in water. Adjust pH to 6.7 with KOH. Add MnCl<sub>2</sub>. Filter to sterilize and keep at 4°C. | ||
− | === | + | ====Tranformation==== |
Add 1µL of plasmids to 50µL of competent cells (make a control tube without plasmid). | Add 1µL of plasmids to 50µL of competent cells (make a control tube without plasmid). | ||
Keep tubes on ice for 30min and heat shock at 42°C for 1min. | Keep tubes on ice for 30min and heat shock at 42°C for 1min. | ||
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Spread cells on Petri dishes in duplicate and incubate at 37°C overnight. | Spread cells on Petri dishes in duplicate and incubate at 37°C overnight. | ||
− | ===Electro-competent cells | + | ===Electro-competent cells=== |
+ | ====Preparation==== | ||
Inoculate 15mL of LB with 200µL of an overnight cell culture. | Inoculate 15mL of LB with 200µL of an overnight cell culture. | ||
Incubate at 37°C and 180rpm until OD<sub>600nm</sub> reaches 0.6. | Incubate at 37°C and 180rpm until OD<sub>600nm</sub> reaches 0.6. | ||
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Wash twice with 10mL of 10% glycerol. | Wash twice with 10mL of 10% glycerol. | ||
Put cells in 200µL of 10% glycerol and use for electroporation. | Put cells in 200µL of 10% glycerol and use for electroporation. | ||
+ | |||
+ | ===Transformation==== |
Revision as of 15:37, 6 July 2016