Erman's Lab:DNA Miniprep with Alkaline Lysis

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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

  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

BioStream version

Following is the Erman's Lab:DNA Miniprep with Alkaline Lysis protocol in BioStream, 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 BioStream (see Source code). More information about BioStream 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

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