Erman's Lab:DNA Miniprep with Alkaline Lysis: Difference between revisions
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==Materials== | ==Materials== | ||
*STE Stock From Stock(for 50ml) Final | *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 | *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% | |||
|- | |||
| dH<sub>2</sub>O | |||
| up to 10ml | |||
| | |||
|} | |||
*ALKALINE LYSIS 3 | |||
{| | |||
|- | |||
! Stock | |||
! From Stock(for 50ml) | |||
! Final | |||
|- | |||
| 5M KAc | |||
| 30ml | |||
| 3M | |||
|- | |||
| Glacial ac ac | |||
| 5.75ml | |||
| 5M acetate | |||
|- | |||
| dH<sub>2</sub>O | |||
| up to 50ml | |||
| | |||
|} | |||
=='''Procedure'''== | =='''Procedure'''== |
Revision as of 12:45, 17 October 2009
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 cold room for 1.5 min.
- Discard supernatant , 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 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 Clonning: A Labratory Manual for further information