Erman's Lab:DNA Miniprep with Alkaline Lysis

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 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

1. o/n grow single colony in 2ml of LB (+ antibiotics)
2. 1.5 ml into eppendorf.
3. Pellet cells at max speed in cold room for 1.5 min.
4. Discard supernatant , leave pellet as dry as possible.
5. Resuspend pellet in 100 μL Alkaline Lysis SLN1,vortex.
6. Add 200μL freshly prepared AL2, mix by inverting 5-6 times(DO NOT vortex!)
7. Put on ice.
8. Add 300μL AL3,invert tubes to mix.
9. Incubate on ice for 5 min.
10. Centrifuge at max speed for 5 min in cold room.
11. Take the supernatant into a new tube.
12. Add 900 isopropanol at RT. Vortex
13. Centrifuge at max speed for 10 min at RT.
14. Rinse the pellet with 1ml 70%EtOH.
15. Centrifuge at max speed for 5 min at RT.
16. Air dry the pellet.
17. Dissolve each in 50μL TE OR H₂O.

Notes

1. Store solutions at 4 degrees and each time freshly prepare the Buffer 2.

References

1. Please refer to Maniatis' Molecular Clonning: A Labratory Manual for further information