Chromosomal DNA isolation from E. coli
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*water bath at 65°C
Revision as of 11:14, 27 October 2010
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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
- 3 M NaOAc
- 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 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
- 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
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