Erman's Lab:DNA Miniprep with Alkaline Lysis: Difference between revisions

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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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==Materials==
==Materials==
*STE Stock From Stock(for 50ml) Final
*STE
TrisCl 1M TrisCl 0.5ml 10mM
{|
NaCl 5M NaCl 1ml 100mM
|-
EDTA 0.5M EDTA 0.1ml 1mM
! Stock
! From Stock(for 50ml)
! Final
|-
| 1M Tris(pH8)
| 0.5ml
| 10mM
|-
| 5M NaCl
| 1ml
| 100mM
|-
| 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
*ALKALINE LYSIS 1
NaOH 5N NaoH 0.4ml 0.2N
{|
SDS 10%SDS 1ml 1%
|-
dH2O Up to 10ml
! 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 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


*ALKALINE LYSIS 2
{|
|-
! Stock
! From Stock(for 10ml)
! Final
|-
| 5N NaOH
| 0.4ml
| 0.2N
|-
| 10% SDS
| 1ml
| 1%
|-
| dH<sub>2</sub>O
| up to 10ml
|
|}
*ALKALINE LYSIS 3
{|
|-
! Stock
! From Stock(for 50ml)
! Final
|-
| 5M KAc
| 30ml
| 3M
|-
| Glacial ac ac
| 5.75ml
| 5M acetate
|-
| dH<sub>2</sub>O
| up to 50ml
|
|}


=='''Procedure'''==
=='''Procedure'''==

Revision as of 12:45, 17 October 2009

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
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