Erman's Lab:DNA Miniprep with Alkaline Lysis
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(New page: ==Overview== Miniprep DNA from E. Coli ==Materials== *STE Stock From Stock(for 50ml) Final TrisCl 1M TrisCl 0.5ml 10mM NaCl 5M NaCl 1ml 100mM EDTA 0.5M EDTA 0.1ml 1mM *ALKALINE LYSIS1 S...)
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Revision as of 15:00, 17 October 2009
Miniprep DNA from E. Coli
- STE Stock From Stock(for 50ml) Final
TrisCl 1M TrisCl 0.5ml 10mM NaCl 5M NaCl 1ml 100mM EDTA 0.5M EDTA 0.1ml 1mM
- ALKALINE LYSIS1 Stock From Stock(for 50ml) Final
TrisCl 1M Tris(pH8) 1ml 20mM EDTA 0.5M EDTA 1ml 10mM Glucose FW=108.2g 0.45g 50mM
- ALKALINE LYSIS 2 Stock From Stock(for 10ml) Final
NaOH 5N NaoH 0.4ml 0.2N SDS 10%SDS 1ml 1% dH2O Up to 10ml
- ALKALINE LYSIS 3 Stock From Stock(for 50ml) Final
KAc 5M KAc 30ml 3M Acetic acid Glacial ac ac 5.75ml 5M acetate dH2O Up to 50ml
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.
- Store solutions at 4 degrees and each time freshly prepare the Buffer 2.
- Please refer to Maniatis' Molecular Clonning: A Labratory Manual for further information