Thursday, December 04, 2003

Description
A 10-min plasmid DNA miniprep method

Procedure
1. Spin down 1.5 ml of E. coli cells in a microfuge (13 Krpm, room temp., 30 to 45 sec) and remove medium

2. Add 50 ml of TEN (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 100 mM NaCl)

3. Resuspend cells using a multivortexer (VWR) (2 min.)

4. Add 50 ml of phenol/chloroform/isoamyl alcohol (25/24/1)

5. Mix briefly using a multivortexer (2 sec.)

6. Spin to release plasmid DNA in a microfuge (room temp., at least 5 min., can be as long as 15 min. when a large number of samples are handled simultaneously)

7. Remove the upper clear layer without touching the interface

8. Add 17ml of 7.5 M NH4OAc and 100 ml of isopropanol (room temp.)

9. Mix well and spin for 1 min. in a microfuge

10. Rinse the pellet with 70% EtOH (1 to 1.5 ml) and dry it completely

11. Resuspend the pellet in 40 ml of H2O. Use 2 ml of DNA for digestion (30 to 60 min in 15 ml with RNase or do an "express" digestion in a microwave oven for 10 sec. three times). Use
18 ml of DNA for sequencing (without RNase, alkaline denaturation works better than DMSO denaturation). Store the DNA at -20oC.




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