Genomic miniprep/Sigma kit

Overview
Protocol for the genomic DNA extraction of Acetobacter xylinum.

Materials

 * Sigma GenElute™ Bacterial Genomic DNA Kit
 * Inoculate of A. xylinum after at least 3 days of growth.

Protocol
See here |SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC for the detailed pdf instructions that come with this kit. This protocol is tailored to the extraction of A. xylinum genomic DNA.
 * 1) Pre-heat water bath to 55°C.
 * 2) Pipette 500 ul of Column preparation solution into each pre-assembled binding column and spin down at 12,000 X g for 1 min. Discard the eluate.
 * 3) Pellet 1.5 ml of an overnight culture of xylinum in an eppendorf tube, centrifuge for 2 minutes at 12,000 X g.
 * 4) Resuspend the pellet thoroughly in 180 ul lysis buffer T.
 * 5) Add 20 ul of Proteinase K solution to the to the resuspension solution. Invert 4-6 times to mix and incubate for 30 minutes at 55°C.
 * 6) Add 200 ul of lysis solution C. Vortex mixture thoroughly for 15 seconds and incubate again at 55°C for 10 minutes.
 * 7) Add 200 ul of ethanol (95-100%) to the lysate and mix thoroughly by vortexing for 5-10 seconds. Ensure mixture is homogeneous.
 * 8) Transfer the cleared lysate into the binding column.
 * 9) Centrifuge at 6500 X g for 1 minute. Replace the collection tube.
 * 10) Add 500 ul of Wash Solution 1 to the column and centrifuge for 1 minute at 6500 X g. Replace the collection tube.
 * 11) Add 500 ul of Wash Solution to the column and centrifuge for 3 minutes at max speed (16,000 X g).
 * 12) Centrifuge the column for an additional minute to remove any excess ethanol.
 * 13) Add 200 ul of elution to the binding column and centifuge for 1 minute at 6500 X g.

Contact

 * morto077@uottawa.ca

or instead, discuss this protocol.

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