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1. Grow O/N in 1.5 ml LMM or Terrific broth (see Reagents) with 75ug/ml Amp
2. Pour into eppendorf tube and spin down cells at 7-8K for 2 min
3. Aspirate s/n and resuspend in 50ul 25mM Tris pH 8, 10 mM EDTA; leave lids open
4. Add 100ul of freshly prepared 1% SDS, 0.2M NaOH (5ml = 100ul 10M NaOH added to 4.4ml DDW then 500ul 10% SDS). Add it forcefully and you don't need to vortex
5. Add 75ul KoAc solution and vortex
6. Add 100ul of phenol/CHI3, close lids, vortex
7. Spin 13K for 2 mins
8. Remove supernatant, add to 500ul ethanol. Vortex and spin at 13 K for 5 min
9. Aspirate s/n, removing all ethanol
10. Resuspend in 50ul TE
11. Digest 2-5ul, adding 1ul preboiled 10mg/ml RNAse A (see Reagents for preparation).
KOAc solution:
60 ml 5M potasium acetate
11.5 ml glacial acetic acid
28.5 ml DDW
NOTE. Large scale plasmid preps can be made quickly by scaling all reagents of the "mini prep" procedure 1000-fold. Once the phenol step has been completed treat the supernatant with 50ul 10mg/ml RNA'se for 1hr, isopropanol ppte (0.6volumes) and resuspend DNA in 400ul of TE. reprecipitate. If the ppte is very cloudy (residual RNA) hook the DNA out (fluffy ball) to separate it from RNA.