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Wednesday, November 19, 2003
Description No Description 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. Recipes Medium and Buffers Psi broth (per liter) compound amount Bacto yeast extract 5 g Bacto Tryptone 20 g magnesium sulfate 5 g pH 7.6 with potassium hydroxide TfbI (per 200 ml) 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 TfbII (per 100 ml) 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 Supplies Tips (责任编辑：泉水) |
Rubidium Chloride method for Transformation Competent E. col
时间:2005-07-18 00:00来源:Scienceboard.net 作者:admin
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