Solutions Procedure

Solution I

1. 50mM glucose
2. 25mM Tris-HCl (pH 8.0)
3. 10mM EDTA (pH 8.0)

Filter sterilization　Cool on ice just before start mini-prep

Solution II

1. 0.2N NaOH
2. 1% SDS

   Fresh preparation. Keep at room temperature

Solution III

　
1. 5M Potassium Acetate 60ml
2. Acetic acid 11.5ml

dH2O 28.5ml

TE containing RNase A

1. 1ml TE
2. 5μl of 10mg/ml RNase A　or 20ｵg/ml RNase A

• Pick up a transformed bacteria colony with sterilized tooth pick and culture in 3ml of LB containing appropriate antibiotics in a test tube.
• Keep 0.5ml of bacteria cultured LB broth as a stock
• Spin down rest of bacteria for 1min at 15000 RPM/4｡C
• Discard the supernatant as much as possible
• Suspend the pellet in 100ｵl of Solution I and mix by pipetting
• Add 200ul of Solution II, mix well by swirling for complete lysis
• Keep on ice for 5min
• Add 150ul of cold Solution III and vortex well
• Keep on ice for 5min
• Spin for 5min at 15,000 RPM/4｡C for pelleting
• Take supernatant (c.a. 0.5ml) and transfer to a new tube
• Add equal volume of phenol/chloroform/isoamyl alcohol
• Take aqueous phase to a new tube
• Add equal volume of chloroform
• Take aqueous phase
• Add equal volume of isopropyl alcohol
• Leave for 2min at room temperature
• Spin for 10 min at 15,000 RPM/room temperature
• Remove sup. 70% EtOH wash
• Dry pellet briefly by speed vac.
• Resuspend in 40ul of TE containing 20ug/ml RNase A

Calculation of DNA Concentration

Dilute the sample solution to one hundredth

Mesure absorbance of DNA solution at 260nm

DNA concentration = A260 X 50 X 100 (ng/ul)
= A260 X 5 (ug/ul)

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