Alkaline Lysis Miniprep protocol
From 2007.igem.org
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- Applications:
- Purification of plasmid DNA from bacterial cultures
- Time to complete protocol:
- Lab time: 3h
- Waiting time: 30min, 15min, 1hour, overnight.
- Approximate cost of materials: $
Contents |
Method from primary and secondary reagents
Primary & secondary Reagents Required including controls
- Bacterial colony
- appropriate antibiotic
- LB (cupboard)
- EDTA
- Glycerol
- TRIS
- NaOH
- SDS
- Potassium Acetate
- Phenol
- Chloroform
- 100% Ethanol
- 70% Ethanol
- milliQ water
- RNAse
Method including controls
- Inoculate 5mL of LB medium with appropriate antibiotic (e.g. 5 microliter of ampicillin) with a single bacterial colony and incubate at 37 degrees C overnight with vigorous shaking.
- Pour 1.5mL of the above culture into an Eppendorf tube. Centrifuge for 1 min. at 10,000g and discard the supernatant by aspiration/decanting.
- Resuspend the pellet by vortexing in 100ul of ice-cold Solution A:
50mM Glucose 10mM EDTA 25mM Tris Cl (pH 8.0)
- Add 200 microliters of freshly prepared Solution B:
0.2M NaOH 1% SDS
invert rapidly 4 times. No vortexing.
- Add 150 microliters of ice-cold Solution C:
3M potassium 5M acetate (pH 4.8)
- vortex for 10 seconds and place on ice for 15 minutes.
- Transfer the supernatant to a fresh tube.
- Add an equal amount (400ul) of phenol/chroloform (200/200ul).
Vortex briefly and centrifuge at 10,000g for 2 minutes.
- Transfer the supernatant to a fresh tube.
- Add two volumes (800ul) of 100% ethanol. Vortex and place on ice for 20 minutes.
Centrifuge at 10,000g for 15 minutes. Discard the supernatant.
- Wash by adding 400ul of 70% ethanol. Centrifuge for 10 minutes.
Discard the supernatant and allow the tube to dry (e.g. vacuum dessicator).
- Add 30ul of TE buffer (or water) with a small amount of RNAase.
Equipement Required
- microcentrifuge: 1.7ml
- Ice box
- Pipettes
- ice
