Overview
Miniprep DNA from E. Coli
Materials
- STE
| Stock | From Stock(for 50ml) | Final |
|---|---|---|
| 1M Tris(pH8) | 0.5ml | 10mM |
| 5M NaCl | 1ml | 100mM |
| 0.5M EDTA | 0.1ml | 1mM |
- ALKALINE LYSIS 1
| Stock | From Stock(for 50ml) | Final |
|---|---|---|
| 1M Tris(pH8) | 1ml | 20mM |
| Glucose FW:108.2 | 0.45g | 50mM |
| 0.5M EDTA | 0.1ml | 10mM |
- ALKALINE LYSIS 2
| Stock | From Stock(for 10ml) | Final |
|---|---|---|
| 5N NaOH | 0.4ml | 0.2N |
| 10% SDS | 1ml | 1% |
| dH2O | up to 10ml |
- ALKALINE LYSIS 3
| Stock | From Stock(for 50ml) | Final |
|---|---|---|
| 5M KAc | 30ml | 3M |
| Glacial ac ac | 5.75ml | 5M acetate |
| dH2O | up to 50ml |
Procedure
Isolation of Mononuclear Cells
- o/n grow single colony in 2ml of LB (+ antibiotics)
- 1.5 ml into eppendorf.
- Pellet cells at max speed in a cold room for 1.5 min.
- Discard supernatant, and leave pellet as dry as possible.
- Resuspend pellet in 100 μL Alkaline Lysis SLN1, vortex.
- Add 200μL freshly prepared AL2, mix by inverting 5-6 times(DO NOT vortex!)
- Put on ice.
- Add 300μL AL3,invert tubes to mix.
- Incubate on ice for 5 min.
- Centrifuge at max speed for 5 min in a cold room.
- Take the supernatant into a new tube.
- Add 900 isopropanol at RT. Vortex
- Centrifuge at max speed for 10 min at RT.
- Rinse the pellet with 1ml 70%EtOH.
- Centrifuge at max speed for 5 min at RT.
- Air dry the pellet.
- Dissolve each in 50μL TE OR H2O.
Notes
- Store solutions at 4 degrees and each time freshly prepare the Buffer 2.
References
- Please refer to Maniatis’ Molecular Cloning: A Laboratory Manual for further information