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...)
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===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 300μL [[TE]] and add 40μL 10%SDS and 3μL 0.5M 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 750μL isopropanole 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
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* resuspend pellet in 500μL TE and add 2μL RNase A (25mg/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 2μL [[proteinase K]] (25mg/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 1ml ethanol and 40μL 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 50μL 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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==Acknowledgments==
 
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Acnkowledge any help you had in development, testing, writing this protocol.
 
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==References==
 
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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]]

Revision as of 07:03, 19 February 2009

Contents

Curators

Torsten Waldminghaus 10:01, 9 December 2008 (EST)

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'.

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