Erman's Lab:DNA Miniprep with Alkaline Lysis

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Contents

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


Procedure

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
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