This plasmid miniprep protocol is suitable for purification of all plasmid DNA for general use (it is not suitable for preparation of DNA for sequencing).
- Grow the bacteria O/N at 37˚C with vigorous shaking in 2ml of L-broth in 10ml glass tubes plus required antibiotic.
- Harvest the cultures by centrifugation in EP tubes (1 minute in Microcentaur) and resuspend in 200µl of lysis buffer (50mM glucose, 25mM Tris.HCl pH8, 10mM EDTA, 4mg/ml lysozyme).
- After 5m at R/T, add 400µl of freshly prepared 0.2M NaOH, 1% SDS, and mix by inverting 3-6 times. NB, The suspension should become clear and viscous.
- After 5m on ice, add 300µl 7.5M ammonium acetate (pH 7.8 without adjustment) and mix the contents of the tube gently for a few seconds.
- Keep the tube at 0˚C for 10m to allow most of the protein, high MW RNA and chromosomal DNA to precipitate.
- Spin at 10K for 5m.
- Remove the clear supernatant, and transfer to another tube.
- Add 0.6vol of isopropanol (500µl) and incubate at R/T for 10m.
- Spin at 15K for 10m.
- Remove the supernatant by aspiration . Add 1ml of 70% ethanol carefully so as not to disturb the pellet, spin for 4 m, then remove the supernatant by aspiration.
- Leave the tubes inverted on a tissue for 15m at R/T.
- Dissolve the pellet in 100µl TE containing 1:100 stock RNAase A (20mg/ml). Incubate on ice for 15m then run samples on a gel (Run 1 or 2ml on an appropriate agarose gel depending on yield. Very poor yields use 3-10ml).
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