"
| Rubidium chloride | 6.00 g |
|---|---|
| Potassium acetate | 2.45 g |
| Calcium chloride * 2 H2O | 0.75 g |
| Glycerol | 75.00 g |
Team:Groningen/Template/MODULE/Notebook/protocols/transformationecoli
From 2014.igem.org
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Revision as of 00:53, 18 October 2014
Transformation of Escherichia coli
Buffers
500 ml RF1 buffer
Adjust the pH to 5.8 using 0.2 M acetic acid. Sterilise by filtration.
300 ml RF2 buffer
Adjust the pH to 6.8 with 1 M NaOH. Sterilise by filtration.
| 0.5 MOPS pH 6.8 | 6.00 ml |
|---|---|
| Rubidium chloride | 0.36 g |
| Calcium chloride * 2 H2O | 3.30 g |
| Glycerol | 45.00 g |
Preparing chemically competent E. coli
Grow an overnight culture E. coli in 5 ml LB, at 37 °C. Inoculate 200 ml of prewarmed LB with 2 ml of the overnight culture the next morning. Grow the cultures until an OD600 of 0.3 - 0.4 is obtained. Put the cells on ice for 15 minutes and then pellet them by centrifuging them for 15 minutes with 2700 rpm at 4 °C. Resuspend the pellets in 16 ml RF1 buffer and leave them on ice for 15 minutes. Centrifuge the cells again for 15 minutes with 2700 rpm at 4 °C. Resuspend the pellet in 4 ml RF2 buffer. Flash freeze aliquots of the cells using liquid nitrogen.
Transformation
Add the DNA to 50 μl chemically competent E. coli. Put the cells on ice for 30 minutes. Give the cells a heatshock by placing them at 42 °C for 45 seconds. Put the tubes back on ice and add 300 ml LB medium. Incubate the cells for an hour at 37 °C. Plate the cells on LB agar.
