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ANNETTE DEICHMANN AND KLAUS DEICHMANN
• Buffer QF (1.25 M NaCl, 50 mM Tris/HCl, 15% ethanol (pH 8.5))
• Isopropanol
• 70% ethanol (ice-cold)
• TE buffer (10 mM Tris; 1 mM EDTA (pH 8.0))
Supplies
• Centrifuge
• Shaker
• Sterile toothpicks
• Spectrophotometer
Procedure
ln advance
1. With a sterile toothpick inoculate 5 ml of LB medium containing the
appropriate antibiotic with a single colony of the transformed E. coli.
Let the cells grow overnight with shaking at 37°C.
2. Inoculate 148.5 ml of LB medium containing the appropriate antibiotic
with 1.5 ml ofthe overnight culture from step 1. Growthe cells with shaking overnight at 37°C.
Standard reaction
3. Harvest the bacterial cells by centrifugation at 4°C and 6000 x g for 15
min. Discard the supernatant.
4. Dissalve 1 mg of RN ase A in 10 ml of buffer P 1 and resuspend the bacterial cells in this solution.
5. Add 10 ml ofbuffer P2 and mix very gently. Incubate at room temperature for 5 min.
6. Add 10 ml of ice-cold buffer P3, mix very gently and incubate on ice for 20
min.
7. Centrifuge at 30,000 x g for 30 min at 4°C. Save the supernatant.
8. Ifthe supernatant is not clear, a second centrifugation (30,000 x g, 4°C,
15 min) is recommended. The turbid supernatant can also be filtered
over a folded filter.
9. Apply 10 ml ofbuffer QBT to a QIAGEN-tip 500 and allow the column to
empty by gravity flow ( equilibration).