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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 08:13, 27 October 2010
, 27 October 2010→Procedure: typo and spaces between numbers and units
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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) | * 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 | * Spin down cells 3 min max. speed at 4°C and discard supernatant | ||
* resuspend in | * resuspend in 300 μL [[TE]] and add 40 μL 10% SDS and 3 μL 0.5 M EDTA | ||
* incubate 5 min at 65°C | * incubate 5 min at 65°C | ||
* add | * add 750 μL isopropanol and mix | ||
* spin at max. speed for 5 min | * spin at max. speed for 5 min | ||
* resuspend pellet in | * resuspend pellet in 500 μL TE and add 2 μL RNase A (25 mg/ml) | ||
* incubate for 30 min at 65°C | * incubate for 30 min at 65°C | ||
* add | * add 2 μL [[proteinase K]] (25 mg/ml) and incubate at 37°C for 15 min | ||
* phenol extract (2x phenol & 2x chlorophorm) | * phenol extract (2x phenol & 2x chlorophorm) | ||
* precipitate over night with | * 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 | * 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== | ||
