DNA转化实验指导
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Rubidium chloride method Inoue "ultra-competent" method
Cosmid packaging protocol
DNA Ligation and Transformation Protocols
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 Cell s 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 Cell s 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 Cell s with 2-10 ul of a ligation reaction, and you should get between 1x10exp8 to 1x10exp9 cfu"s/ug DNA.
Medium and Buffers
Psi broth (per liter) compoundamountBacto yeast extract5 gBacto Tryptone20 g magnesium sulfate5 g pH 7.6 with potassium hydroxide
TfbI (per 200 ml) compoundamountfinal molarity/conc.potassium acetate.588 g30 mM rubidium chloride2.42 g100 mMcalcium chloride0.294 g10 mMmanganese chloride2.0 g50 mMglycerol30 ml15% v/vpH 5.8 with dilute acetic acid
TfbII (per 100 ml) compoundamountfinal molarity/conc.MOPS0.21 g10 mMcalcium chloride1.1 g75 mMrubidium chloride0.121 g10 mMglycerol15 ml15% v/vpH 6.5 with dilute NaOH
Transformation "Ultra-Competent" E. coli (Inoue Method)
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Inoue, H., H. Nojima, and H. Okayama. 1990. High efficiency transformation of Escherichia coli with plasmids. Gene 96 :23-28. Citation Abstract
