Team:Mexico-UNAM-CINVESTAV/Protocols

From 2010.igem.org

Alkaline Lysis Mini Plasmid Preps

1. Grow O/N in 1.5 ml LMM or Terrific broth (see Reagents) with 75µg/ml Amp
2. Pour into ependorf tube and spin down cells at 7-8K for 2 min

3. Aspirate s/n and resuspend in 50µl 25mM Tris pH 8, 10 mM EDTA; leave lids open 4. Add 100µl of freshly prepared 1% SDS, 0.2M NaOH (5ml = 100ul 10M NaOH added to 4.4ml DDW then 500µl 10% SDS). Add it forcefully and you don't need to vortex 5. Add 75µl KoAc solution and vortex 6. Add 100µl of phenol/CHI3, close lids, vortex 7. Spin 13K for 2 mins 8. Remove supernatant, add to 500µl ethanol. Vortex and spin at 13 K for 5 min 9. Aspirate s/n, removing all ethanol 10. Resuspend in 50µl TE 11. Digest 2-5ul, adding 1µl preboiled 10mg/ml RNAse A (see Reagents for preparation). KOAc solution: - 60 ml 5M potassium acetate - 11.5 ml glacial acetic acid - 28.5 ml DDW