"
Team:Hannover/Protocols/Transformation/E.coli Heatshock
From 2014.igem.org
Protocols / Preparation and Transformation of Heatshock Competent E. coli Cells
Preparation of Heatshock competent E. coli cells
Material: |
| Centrifuge |
| 100 mM CaCl2 |
| 86 % glycerin |
Protocol:
After E. coli cell cultures were grown to an optical density (OD600) of ~ 0.3 to 0.4, they were centrifuged for 10 min at 5400 x g and 4 °C. While the supernatant was decanted, the precipitated bacteria had to be resuspended in half the volume which was used for the starting culture by adding 100 mM CaCl2. The suspension was placed on ice for 20 min. After centrifuging with the same settings a second time, only one tenth the volume of 100 mM CaCl2 of what was previously used was added again. After another hour on ice, the E. coli cells were heat shock competent. Depending on the strain, long-term storage must be tested. For that purpose, 86 % glycerin was added up to a total concentration of 15 % (v/v). Before storage at -80 °C, the cultures were properly mixed.
Transformation of Heatshock competent E. coli cells
Material: |
|
| watherbath | |
| shacking and stationary incubator | |
| antibiotics | |
| SOC-medium | 937.5 μl SOB media, 12.5 μl 2 M MgCl2, 50.0 μl 20 % (w/v) glucose |
| SOB-medium | 2.00 % (w/v) Tryptone, 0.50 % (w/v) Yeast extract, 0.06 % 10 mM NaCl, 0.02 % 2,5 mM KCl |
| 2TY media | 1.6 % (w/v) Peptone, 1.0 % (w/v) Yeast extract, 0.5 % (w/v) NaCl ; pH 7 |
Protocol:
Approximately 3-20 μl of ligation product was pipetted into 50 μl of freshly prepared heatshock competent E. coli cells. The mixture stayed on ice for 20 min, before the heatshock was conducted for 45 s at 42 °C. Afterwards 500 μl SOC media was added and the transformed bacteria were cultivated for 45 min shaking with 180 rpm at 37 °C. Subsequently, the bacteria were grown on top of solid, antibiotics containing 2TY media. To allow optimal colony growth, the medium was stored at 37 °C overnight.
