Erman's Lab:DNA Miniprep with Alkaline Lysis
From OpenWetWare
Jump to navigationJump to search
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
BioCoder version
Following is the Erman's Lab:DNA Miniprep with Alkaline Lysis protocol in BioCoder, a high-level programming language for expressing biology protocols. What you see here is the auto-generated text ouput of the protocol that was coded up in BioCoder (see Source code). More information about BioCoder can be found on my home page. Feel free to mail me your comments/ suggestions.Vaishnavi
Text Output
Erman's Lab:DNA Miniprep with Alkaline Lysis protocol
Source Code
Erman's Lab:DNA Miniprep with Alkaline Lysis protocol - source code