Chromosomal DNA isolation from E. coli

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(New page: ==Curators== '''~~~~''' ''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 com...)
Current revision (10:16, 31 July 2012) (view source)
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{{back to protocols}}
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==Curators==
==Curators==
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'''[[User:Torsten Waldminghaus|Torsten Waldminghaus]] 10:01, 9 December 2008 (EST)'''
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'''[[User:Torsten Waldminghaus|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 [[OpenWetWare:Information management/Protocol curators|discussing the idea of protocol curators]].  Please contribute.''
''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 [[OpenWetWare:Information management/Protocol curators|discussing the idea of protocol curators]].  Please contribute.''
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==Materials==
==Materials==
*10% SDS
*10% SDS
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*Isopropanole
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*Isopropanol
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*Ethanole
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*Ethanol
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*3M Na-acetate
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*3 M NaOAc
===Reagents===
===Reagents===
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Biological resources e.g. cell lines, buffers (link to a method for making them), enzymes, chemicals, kits, etc.
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*[[TE]]
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*[[Killing Buffer]]
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===Equipment===
===Equipment===
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Any equipment used to perform the protocol (link to a method for using them).
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*water bath at 65°C
==Procedure==
==Procedure==
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A step by step guide to the experimental procedure.
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* grow culture in your favorite medium
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* Mix samples directly with ice cold [[Killing Buffer]] in ratio 1:1 and put on ice (samples should be processed as fast as possible)
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If you find it helpfull you could use the following icons to highlight important parts:
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* Spin down cells 3 min max. speed at 4°C and discard supernatant
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* resuspend in 300 μL [[TE]] and add 40 μL 10% SDS and 3 μL 0.5 M EDTA
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[[Image:Difficult step.png]] icon to highlight difficult steps
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* incubate 5 min at 65°C
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* add 750 μL isopropanol and mix
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[[Image:Critical step.png]] warn about critical steps
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* spin at max. speed for 5 min at room temperature and discard supernatant
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* resuspend pellet in 500 μL TE and add 2 μL RNase A (25 mg/ml)
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[[Image:Time required.png]] help people plan how long steps will take
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* incubate for 30 min at 65°C
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* add 2 μL [[proteinase K]] (25 mg/ml) and incubate at 37°C for 15 min
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[[Image:Pause point.png]] where protocol can be interrupted
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* phenol extract (2x phenol & 2x chlorophorm)
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* precipitate over night with 1 ml ethanol and 40 μL 3M Na-Acetate
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[[Image:Optional step.png]] steps that can be omitted or included
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* spin down DNA at 4°C for 15 min, wash in 70% ethanol and resuspend pellet in 50 μL dH<sub>2</sub>O
==Critical steps==
==Critical steps==
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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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It might also be good to add an image to show the workflow and timescales for experiment planning.
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==BioCoder version==
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Following is the Chromosomal DNA isolation from E. coli 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]]
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==Acknowledgments==
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====Text Output====
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Acnkowledge any help you had in development, testing, writing this protocol.
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[[Chromosomal DNA isolation from E. coli protocol]]
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====Source Code====
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==References==
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[[Chromosomal DNA isolation from E. coli protocol - source code]]
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See [[OpenWetWare:Biblio]] for information on how to reference within a wiki.
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==Specific Protocols==
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Add links to all the OWW protocols that have been used in making the consensus.
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==Discussion==
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You can [[Talk:{{PAGENAME}}|discuss this protocol]].
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Tag this page with categories to allow easier indexing and searching.  See [[Categories]] for information on existing categories.
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[[Category:Protocol]]
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[[Category:Protocol]][[Category:Needs attention]]

Current revision

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
  • Isopropanol
  • Ethanol
  • 3 M NaOAc

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 300 μL TE and add 40 μL 10% SDS and 3 μL 0.5 M EDTA
  • incubate 5 min at 65°C
  • add 750 μL isopropanol and mix
  • spin at max. speed for 5 min at room temperature and discard supernatant
  • resuspend pellet in 500 μL TE and add 2 μL RNase A (25 mg/ml)
  • incubate for 30 min at 65°C
  • add 2 μL proteinase K (25 mg/ml) and incubate at 37°C for 15 min
  • phenol extract (2x phenol & 2x chlorophorm)
  • precipitate over night with 1 ml ethanol and 40 μL 3M Na-Acetate
  • spin down DNA at 4°C for 15 min, wash in 70% ethanol and resuspend pellet in 50 μL 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'.

BioCoder version

Following is the Chromosomal DNA isolation from E. coli 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

Chromosomal DNA isolation from E. coli protocol

Source Code

Chromosomal DNA isolation from E. coli protocol - source code

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