Erman's Lab:DNA Miniprep with Alkaline Lysis

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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 15:45, 17 October 2009

Contents

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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