Genomic miniprep/Sigma kit

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(New page: ==Overview== Protocol for the genomic DNA extraction of ''Acetobacter xylinum''. =='''Materials'''== *Sigma GenElute™ Bacterial Genomic DNA Kit *Inoculate of A. xylinum after at least...)
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Revision as of 14:26, 18 October 2009

Contents

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 [1] 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 miz 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.

Notes

All questions, input and feedback are welcome!

  1. To pellet the A.xylinum inoculate, the cellulose must be removed from the media. This can be achieved by briefly vortexing the media to break up the cellulose matrix and then removing the cellulose chunks with a pipette. You will see the cells congregating in the liquid media that is left over after cellulose removal.
  2. It is highly recommended that the elution buffer be heated to around 50-60°C before the elution step of the procedure. Also, it is a good idea to warm up the binding column/collection tube as the elution solution is added in a heat block. Let the elution solution incubate for at least 3-5 mins in the heat block before the final spin down. This will improve yield slightly.
  3. Be sure to use a wide-bore pipette tip when transferring the lysate to the pre-prepared binding column.
  4. It is also recommended that a second elution of 200 ul is performed after the initial elution of 200 ul, this will improve yield greatly.
  5. The Proteinase K solution should not be stored in the fridge, keep it stored in the -20°C freezer.
  6. For more information, please visit [2]

Contact

  • morto077@uottawa.ca

or instead, discuss this protocol. -->

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