From 2013.igem.org

Revision as of 05:59, 14 July 2013

Tips for 10 min plasmid DNA minipreparation

1. Grow 2.0 ml overnight culture
2. Spin down 1.0 ml if E. coli overnight cells in a microcentrifuge (13,000 rpm, room temp., 30-45 sec) and remove medium.
3. Add 50 ul of TEN (10 mM Tris-HCl, pH8.0, 1 mM EDTA, 100mM NaCl)
4. Vortex for 2 min. (VWR multivortexer)
5. Add 70 ul phenol/chloroform/isoamyl alcohol (25/24/1, with 1% β-ME)
6. Vortex for 2-3 sec, microcentrifuge for 10 min (room temp.)
7. Transfer upper solution 70 ul (without touching the interface) to new microcentrifuge tube
8. Add 20 ul of NH₄OAc (7.5M) and 100 ul of isopropanol (room temp.)
9. Vortex for 5 sec, microcentrifuge for 1 min (room temp.)
10. Wash the pellet with 70 % EtOH (1 ml) and dry it completely.
11. Resuspend the pellet in 15 ul of dd H₂O or TE. and 1 / 50 RNase. Store at -20^oC

Note. Use 4 ul of DNA for digestion (30-60 min in 15 ul with RNase or “express” digestion 3 X 10 sec in a microwave oven), use 18 ul for sequencing.