DNA转化实验指导
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- Rubidium chloride method
- Inoue "ultra-competent" method
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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 |
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1. Inoculate from an overnight grown in LB.
2. Grow in 250 ml "SOB" at 18C until OD600 = 0.6.
2. On ice for 10 minutes.
3. Spin at 2500 x g (5000 rpm in a Sorvall GSA or 3000 rpm in a Beckman J-6B centrifuge) for 10 min. at 4C.
4. Resuspend cells gently in 80 ml of ice cold "TB". 5. On ice for 10 minutes.
6. Spin at 2500 x g (5000 rpm in a Sorvall GSA, 5500 rpm in a Sorvall SS-34, or 3000 rpm in a Beckman J-6B centrifuge) for 10 min. at 4C.
7. Resuspend cells gently in 20 ml of ice cold "TB".
8. Add DMSO to a final concentration of 7%.
9. Place on ice for 10 minutes.
10. Aliquot into 1-2 ml and freeze in liquid nitrogen.
11. Store in liquid nitrogen.
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Cosmid Cloning: Cell preparation, DNA packaging, and Cell Transfection
Protocol taken from Stratagene's Gigapack packaging extracts instruction manual
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