Chromosomal DNA isolation from E. coli
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Revision as of 11:47, 23 July 2010
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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.
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.
- 10% SDS
- 3M Na-acetate
- water-bath at 65°C
- 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
Referenced from the main protocol, a more thorough explanation of particularly important steps in the protocol.
Referenced from the main protocol, an explanation of what can cause things to go wrong with the protocol.
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'.
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