Section on Neural Gene Expression

Miniprep DNA Preparation

(as provided by Alice Young)

SOLUTIONS:

• Resuspension buffer:
  25mM Tris-HCl (ph8)
  50mM Na₂EDTA
  1% glucose
  Autoclave and store at 4°C.

• NaOH/SDS: 0.2 N NaOH/1% SDS. Prepare fresh for maximum yield.

• 7.5 Ammonium acetate: filtered, not pH'd, and stored at room temp.

• TE: 10mM Tris-HCl, pH8/1mM Na₂EDTA

• 70% Ethanol, room temp.

• 95% Ethanol, room temp.

PROCEDURE:

1. Spin 1-1.5ml of overnight culture at 6000rpm for 6 min. in a microfuge and resuspend pellets in 100µl cold resuspension buffer.
   
   
2. Add 200µl NaOH/SDS. Mix, do not vortex, and place on ice for 15min.
   
   
3. Add 150µl 7.5M ammonium acetate. Mix and place on ice for 15min.
   
   
4. Centrifuge at 14,000rpm in a microfuge for 15min. at room temperature.
   
   
5. Transfer supernatant to a fresh tube containing 900µl 95% etahnol. Mix well and place on ice for 15min. Centrifuge at 14,000rpm for 15min.
   
   
6. Drain the tubes. Rinse pellet with 1ml 70% ethanol, do not mix, and centrifuge for 2min at 14,000rpm.
   
   
7. Drain tubes. Respin for 10sec. Pipet off all liquid. Air dry 30min or vacuum dry 5min. Resuspend in 50µl TE. Solution will be approximately 100ng/µl.
   
   

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