https://gcat.davidson.edu/GcatWiki/index.php?action=history&feed=atom&title=Isolation_of_Genomic_DNA_from_Bacteria 2026-05-17T16:04:29Z Revision history for this page on the wiki MediaWiki 1.28.2 https://gcat.davidson.edu/GcatWiki/index.php?title=Isolation_of_Genomic_DNA_from_Bacteria&diff=11644&oldid=prev 2010-07-15T20:21:32Z

<p></p> <p><b>New page</b></p><div>'''Isolation of Genomic DNA from Bacteria'''<br /> <br /> # Grow 1.5 ml culture of bacteria overnight<br /> # Spin 1 minute to collect cells, pour off supernatant<br /> # Resuspend pellet in 400 ul TE<br /> # Add 50 ul 10% SDS and 50 ul proteinase K (10 mg/ml)<br /> # Incubate 1 hour at 37 C with occasional shaking<br /> # Siphon up and down through 26 G needle<br /> # Add 500 ul saturated phenol:chloroform with isoamyl alcohol (1:24)<br /> # Vortex and spin 1 minute<br /> # Transfer upper aqueous phase to a new tube<br /> # Repeat phenol:chloroform extraction<br /> # Extract twice with chloroform/isoamyl alcohol (24:1)<br /> # Add TE to final aqueous phase to adjust volume to 500 ul<br /> # Add 50 ul 2.5 M NaCl and 1 ml absolute ethanol<br /> # Spin 10 minutes, pour off supernatant<br /> # Resuspend pellet in 100 ul TE, add 5 ul RNase A (5 mg/ml)<br /> # Incubate 30 minute at 37 C<br /> # Add 40 ul 5M sodium acetate and 250 ul isopropanol<br /> # Place in freezer 10 minutes<br /> # Spin 10 minutes<br /> # Wash pellet with 500 ul 70% ethanol<br /> # Allow pellet to dry<br /> # Dissolve pellet in 100 ul of TE or less<br /> # Measure absorbance at 260 nm - typical yield is 4 ug to 10 ug</div>

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