Plasmid miniprep
Plasmid miniprep
Material
- LB plate with antibiotic (4C)
- LB medium with antibiotic (4C)
See Plasmid.
- GTE (50 mM glucose/25 mM Tris-HCl pH 8.0/10 mM EDTA)
- 0.2 N NaOH/1% SDS
- 4 M potassium acetate (4C)
- 5 M LiCl
Day 1
- Pick up E. coli colony by yellow tip.
- Stamp it on new LB plate to make copy plate for midiprep.
- Put tip into 2 ml LB medium in 5 ml tube for miniprep.
- Incubate plate at 37C for o/n.
- Culture tube at 250 rpm at 37C for o/n.
Day 2
- Move 1 ml cultured LB medium to 1.5 ml tube.
- Centrifuge at 13000 rpm at RT for 1 min.
- Suck supernate.
- Add 100 ul GTE.
- Suspend by vortex.
- Add 200 ul 0.2 N NaOH/1% SDS.
- Invert to mix.
- Add 150 ul 4 M potassium acetate.
- Invert to mix.
- Add 450 ul 5 M LiCl.
- Invert to mix.
- Incubate on ice for 5 min.
- Centrifuge at 13000 rpm at RT for 10 min.
- Move 800 ul supernate to new 1.5 ml tube.
- Add 700 ul isopropanol.
- Invert to mix.
- Centrifuge at 13000 rpm at RT for 10 min.
- Remove supernate by decantation.
- Add 500 ul 70% EtOH.
- Centrifuge at 13000 rpm at RT for 5 min.
- Remove supernate by 200 ul pipet.
- Dry up.
- Dissolve in 20 ul H2O.
- Use 5 ul for restriction enzyme cutting.
