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ANNETTE DEICHMANN AND KLAUS DEICHMANN
• Tetracycline, 15 mg/ml in 50% ethanol, sterile-ftltered
Supplies • Autoclave
• Petri dishes
Casting plates
1. Prepare LB Medium:
HzO
Bacto-tryptone (1%)
Bacto-yeast extract (0.5%)
NaCl (1%)
Adjust pH to 7.3 with 1M NaOH
Add H20 to make 1 liter.
950 ml
10 g
Sg
10 g
2. Add 15 g agar to 1liter of LB-medium.
3. Sterilize in the autoclave, cool to 50°C.
4. Add antibiotics if desired:
- 1 ml of a 50 mg/ml solution of ampicillin
- 1 ml of a 15 mg/ml solution of tetracycline
5. Disperse carefully avoiding air bubbles.
6. Pour into Petri dishes. Stack one upon the other and let cool to solidify.
Use approximately 25 ml of LB agar per 100 mm Petri dish.
7. Store the plates at 4°C until used.
Transformation
Reagents • Recombinant plasmid
• LB agar plates containing tetracycline and ampicillin (see above)
• A colony of HB101
Salutions • LB medium (see above)
• 50 mM CaCh (sterile and ice-cold)
• Glycerol sterilized by autoclaving (ice-cold)
Supplies • Shaker or shaking water bath