Transformation in E.coli
From 2007.igem.org
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| - | + | '''Transformation of plasmid DNA to competent E. Coli cells''' | |
| - | + | ''Material and Reagents'' | |
1. SOC | 1. SOC | ||
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10mM MgSO4 | 10mM MgSO4 | ||
10mM MgCl2 | 10mM MgCl2 | ||
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2. 1.5 mL microfuge tubes | 2. 1.5 mL microfuge tubes | ||
3. 42° C waterbath | 3. 42° C waterbath | ||
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5. 37° C shaker | 5. 37° C shaker | ||
| - | Protocol | + | ''Protocol'' |
1. Thaw competent cells on ice. 20–200µL per tube | 1. Thaw competent cells on ice. 20–200µL per tube | ||
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6. Place the tubes immediately on ice for at least 2 min | 6. Place the tubes immediately on ice for at least 2 min | ||
7. Add 250µL of SOC medium to each tube | 7. Add 250µL of SOC medium to each tube | ||
| - | 8. Incubate for 1 hour at 37°C and shake vigorously | + | 8. Incubate for 1 hour at 37°C and shake vigorously |
| - | + | 10. Plate out the suspension on a LB agar plate containing the appropriate antibiotic. | |
| - | + | 11. Incubate the plates overnight at 37°C | |
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| + | [[Image:PlateE.coli.jpg]] | ||
Latest revision as of 15:28, 20 September 2007
Transformation of plasmid DNA to competent E. Coli cells
Material and Reagents
1. SOC
2% Tryptone
0.5% Yeast Extract
10mM NaCl
10mM MgSO4
10mM MgCl2
2. 1.5 mL microfuge tubes
3. 42° C waterbath
4. Ice
5. 37° C shaker
Protocol
1. Thaw competent cells on ice. 20–200µL per tube
2. Add max. 20µL of a ligation reaction
3. Mix very gently!
4. Incubate the tubes on ice for 30 min
5. Heat shock the cells for 30 sec at 42°C
6. Place the tubes immediately on ice for at least 2 min
7. Add 250µL of SOC medium to each tube
8. Incubate for 1 hour at 37°C and shake vigorously
10. Plate out the suspension on a LB agar plate containing the appropriate antibiotic.
11. Incubate the plates overnight at 37°C
