Paul.xu at 16:14, 6 March 2015

2015-03-06T16:14:10Z

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== Protocol 1 ==

Centrifuge 3mL of an overnight culture grown in LB to pellet cells. Resuspend in 0.75mL 1% NaCl. Centrifuge. Resuspend pellet in 0.75mL TES (10mM Tris-HCl, 10mM EDTA, pH 8.0, 2% SDS) Incubate at 75oC for 15 minutes. Vortex. Add 1ml phenol:chloroform (3:1 v/r). Centrifuge. Pipet off the aqueous layer (top) into a fresh tube. Add 1mL chloroform. Centrifuge. Pipet off the aqueous layer (top) into a fresh tube. Add 100μL 3M NaOAc (pH 5.2) & 1mL isopropanol. Invert tube & centrifuge. Add 500μL absolute ethanol & spool out DNA into fresh tube. Resuspend DNA (spooled) in 150 uL TE (10mM Tris-HCl, 2mM EDTA, pH 8.0) with 1μL 50ug/mL RNase. 

 

== Protocol 2 ==

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 at room temperature and discard supernatant 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 

[[Category:Protocols]]

Isolate Genomic DNA from E coli - Revision history

Paul.xu at 16:14, 6 March 2015