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Team:Warsaw/Calendar-Main/21 July 2009
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(New page: {{WarNotebook}} <!-- do not edit above me! --> <html> <h3>Transformation of E. coli with pKS/hly plasmid</h3> <h4>Kama</h4> <ul> <li>chemocompetent E. coli dH5α were incubated on ice...) |
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<li>chemocompetent E. coli dH5α were incubated on ice for 15 minutes</li> | <li>chemocompetent E. coli dH5α were incubated on ice for 15 minutes</li> | ||
| - | <li>ligation mixture was added</li> | + | <li>15μl of ligation mixture was added (and 15μl of control ligation, without insert)</li> |
<li>bacteria were incubated with DNA on ice for 30 minutes</li> | <li>bacteria were incubated with DNA on ice for 30 minutes</li> | ||
<li>heat shock was conducted (1 minute 42°C)</li> | <li>heat shock was conducted (1 minute 42°C)</li> | ||
<li>bacteria were incubated on ice for 3 minutes</li> | <li>bacteria were incubated on ice for 3 minutes</li> | ||
| - | <li>After the heat shock | + | <li>After the heat shock 800μl of SOB medium was added</li> |
<li>Mixture with bacteria was incubated for 1 hour in 37°C</li> | <li>Mixture with bacteria was incubated for 1 hour in 37°C</li> | ||
<li>100μl of mixture was plated on medium containing ampicillin, X-Gal and IPTG</li> | <li>100μl of mixture was plated on medium containing ampicillin, X-Gal and IPTG</li> | ||
Revision as of 22:36, 21 July 2009
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Transformation of E. coli with pKS/hly plasmid
Kama
- chemocompetent E. coli dH5α were incubated on ice for 15 minutes
- 15μl of ligation mixture was added (and 15μl of control ligation, without insert)
- bacteria were incubated with DNA on ice for 30 minutes
- heat shock was conducted (1 minute 42°C)
- bacteria were incubated on ice for 3 minutes
- After the heat shock 800μl of SOB medium was added
- Mixture with bacteria was incubated for 1 hour in 37°C
- 100μl of mixture was plated on medium containing ampicillin, X-Gal and IPTG
- The rest of mixture was rotated for 1 minute, pelet was
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