Chromosomal DNA isolation from E. coli

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Anecdotal observations that might be of use to others can also be posted here; e.g. 'my cells were still floating'.<br>
Anecdotal observations that might be of use to others can also be posted here; e.g. 'my cells were still floating'.<br>
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==BioStream version==
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Following is the Chromosomal DNA isolation from E. coli 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.[[User:Vaishnavi Ananth|Vaishnavi]]
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====Text Output====
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[[Chromosomal DNA isolation from E. coli protocol]]
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====Source Code====
 +
[[Chromosomal DNA isolation from E. coli protocol - source code]]
[[Category:Protocol]][[Category:Needs attention]]
[[Category:Protocol]][[Category:Needs attention]]

Revision as of 02:04, 1 October 2009

back to protocols

Contents

Curators

Torsten Waldminghaus

Anyone should feel free to add themselves as a curator for this consensus protocol. You do not need to be a curator in order to contribute. The OpenWetWare community is currently discussing the idea of protocol curators. Please contribute.

Abstract

This protocol describes the isolation of chromosomal DNA from E. coli cells. The DNA could be used for Southern Blots, as template for PCR or for DNA microarrays.

Materials

  • 10% SDS
  • Isopropanole
  • Ethanole
  • 3M Na-acetate

Reagents

Equipment

  • water-bath at 65°C

Procedure

  • grow culture in your favorite medium
  • Mix samples directly with ice cold Killing Buffer in ratio 1:1 and put on ice (samples should be processed as fast as possible)
  • Spin down cells 3 min max. speed at 4°C and discard supernatant
  • resuspend in 300μL TE and add 40μL 10%SDS and 3μL 0.5M EDTA
  • incubate 5 min at 65°C
  • add 750μL isopropanole and mix
  • spin at max. speed for 5 min
  • resuspend pellet in 500μL TE and add 2μL RNase A (25mg/ml)
  • incubate for 30 min at 65°C
  • add 2μL proteinase K (25mg/ml) and incubate at 37°C for 15 min
  • phenol extract (2x phenol & 2x chlorophorm)
  • precipitate over night with 1ml ethanol and 40μL 3M Na-Acetate
  • spin down DNA at 4°C for 15 min, wash in 70% ethanol and resuspend pellet in 50μL dH2O

Critical steps

Referenced from the main protocol, a more thorough explanation of particularly important steps in the protocol.

Troubleshooting

Referenced from the main protocol, an explanation of what can cause things to go wrong with the protocol.

Notes

Referenced from the main protocol, any comments about the protocol should be made here; i.e. how it was developed. Any comments added should be signed (by adding *'''~~~~''': in front) and explained. Links to FAQs/tips provided by other sources such as the manufacturer or other websites would be best made here.
Anecdotal observations that might be of use to others can also be posted here; e.g. 'my cells were still floating'.

BioStream version

Following is the Chromosomal DNA isolation from E. coli 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

Chromosomal DNA isolation from E. coli protocol

Source Code

Chromosomal DNA isolation from E. coli protocol - source code

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