CONTENT
Rubidium Chloride method for Transformation Competent E. coli
Procedure
1. Inoculate 1 ml from overnight culture into 100 ml Psi broth (scale up or down as needed). Incubate at 37 C with aeration to A550=0.48
2. Ice 15 min.
3. Pellet cells in appropriate centrifuge tube 3-5000 x g 5 min (~5000 rpm in a Sorvall SS-34 rotor)
4. Discard supernatant and add 0.4 volume (ie of original volume, here it is 40 ml) TfbI, resupend and ice 15 min.
5. Pellet cells as in #3.
6. Discard supernatant and resupend in 0.04 volume TfbII, ice 15 min and either use immediately or quick freeze at -70C for storage. I usually save these in 0.25 to 0.5 ml aliquots. Quick freeze in ethanol-dry ice or liquid nitrogen prior to storage in a -70 to -80 C freezer. Thaw on ice just before using in a transformation experiment.
I typically transform 50 ul cells with 2-10 ul of a ligation reaction, and you should get between 1x10exp8 to 1x10exp9 cfu's/ug DNA.
Medium and Buffers
| compound | amount |
|---|---|
| Bacto yeast extract | 5 g |
| Bacto Tryptone | 20 g |
| magnesium sulfate | 5 g |
| pH 7.6 with potassium hydroxide | |
| compound | amount | final molarity/conc. |
|---|---|---|
| potassium acetate | .588 g | 30 mM |
| rubidium chloride | 2.42 g | 100 mM |
| calcium chloride | 0.294 g | 10 mM |
| manganese chloride | 2.0 g | 50 mM |
| glycerol | 30 ml | 15% v/v |
| pH 5.8 with dilute acetic acid | ||
| compound | amount | final molarity/conc. |
|---|---|---|
| MOPS | 0.21 g | 10 mM |
| calcium chloride | 1.1 g | 75 mM |
| rubidium chloride | 0.121 g | 10 mM |
| glycerol | 15 ml | 15% v/v |
| pH 6.5 with dilute NaOH | ||
Transformation "Ultra-Competent" E. coli (Inoue Method)
- Inoue, H., H. Nojima, and H. Okayama. 1990. High efficiency transformation of Escherichia coli with plasmids. Gene 96:23-28. Citation Abstract
