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Chromosomal DNA isolation from E. coli: Difference between revisions
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Chromosomal DNA isolation from E. coli (view source)
Revision as of 04:03, 19 February 2009
, 19 February 2009no edit summary
(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=== | ||
*[[TE]] | |||
*[[Killing Buffer]] | |||
===Equipment=== | ===Equipment=== | ||
*water-bath at 65°C | |||
==Procedure== | ==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 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> | ||
[[Category:Protocol]] | [[Category:Protocol]][[Category:Needs attention]] | ||
