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A. Adjust to pH 5.8 with 0.2 M acetic acid (do not adjust pH with KOH).
B. Add ddH2 O to 200 ml. Filter sterilize. Store refrigerated at 4°C
2. Pick a single fresh colony the next day, inoculate 5 mls of YETM medium and grow O/N at 37°C.
• DO NOT vortex cells at any time after this point in the procedure.
3. Dilute 1 ml of O/N culture into 50 mls YETM medium prewarmed to 37°C, and grow at 37°C for 2h
with agitation. Volumes can be scaled up 5X and all of the 5 ml O/N culture can be used.
4. Chill 5 minutes on ice and spin in IEC centrifuge for 10 minutes at 2000 rpm at 4°C. Prechill the
centrifuge to 4°C. Prechill centrifuge tubes on ice.
• DO NOT allow the cells warm up over 4°C at any time in the procedure.
5. Immediately aspirate off all of the supernatant. Resuspend the cells in 10 ml of ice-cold TFB1 with
gentle re-pipetting. Use chilled pipette(s).
6. Incubate cells for 5 minutes on ice. Spin in IEC centrifuge for 10 minutes at 2000 rpm at 4°C.
7. Immediately aspirate off all of the supernatant. Resuspend the cells in 2.0 ml of ice-cold TFB2
with gentle re-pipetting. Use chilled pipette or pipette tip(s).
8. Incubate cells on ice for 15 minutes. Cells may be used for transformation now (next page), or
frozen as described below.
To freeze: aliquot cells in 220 µl volumes into prechilled 0.5 ml microfuge tubes (on ice). Freeze
immediately on dry ice. Store cells frozen at -80°C.
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III. Transformation of Competent Cells
1. Thaw cells at room temperature until just thawed and then place on ice for 10 minutes.
• In general, the maximum amount of DNA that should be used is 0.1 volume of the cells. Add no
more than 100 ng of DNA.
7. Add 1 ml of YETM medium and incubate at 37°C for 45 minutes to 1 hour, with slow gentle shaking.
• For blue/white color selection, spread IPTG and X-gal on plates now. Place at 37°C until use.
8. Plate 0.1 - 0.2 ml of the transformed cells on LB-plates containing the appropriate antibiotic,
with IPTG and X-gal if needed. Incubate overnight at 37°C. Place at 4°C to store and/or enhance
blue color.
1. Transform 100 µl of cells with 1 µl (10 pg) of pUC19 monomer (0.01 µg/µl).
3. Efficiency = # of colonies X 4 X 105 = # of colonies per µg. Should obtain 1-5 X 107 /µg after one
freeze-thaw cycle.
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