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'''==
Line 52: Line 114:
==References==
==References==
#Please refer to Maniatis' Molecular Clonning: A Labratory Manual for further information
#Please refer to Maniatis' Molecular Clonning: A Labratory Manual for further information
==BioCoder version==
Following is the Erman's Lab:DNA Miniprep with Alkaline Lysis protocol in BioCoder, 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 BioCoder (see Source code). More information about BioCoder can be found on my home page. Feel free to mail me your comments/ suggestions.[[User:Vaishnavi Ananth|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]]




Latest revision as of 02:52, 19 November 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

BioCoder version

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