Erman's Lab:DNA Miniprep with Alkaline Lysis

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

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