Team:Calgary/Notebook/Protocols/Process24
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Revision as of 02:16, 29 September 2011
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Genomic DNA Isolation from Pseudomonas spp.
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This protocol is used to isolate genomic DNA (gDNA) from Pseudomonas spp. (and any gram negative bacteria), and is adapted from the Chen & Kuo (1993) publication in Nucleic Acids Research.
Reagents
Protocol
- Centrifuge 1.5mL of a saturated culture for 3 min at 12000 rpm.
- Decant the supernatant, and resuspend the cell pellet in 200µL of lysis buffer. Mix by vigorous pipetting.
- Add 66µL of 5M NaCl to remove proteins and cell debris. Mix well.
- Centrifuge at 12000rpm for 10 min at 4°C.
- Transfer the clear supernatant to a new tube, and precipitate DNA by adding 100% ethanol, and incubating at -20°C for at least 1 hour.
- Centrifuge for 10 minutes at 4°C.
- Decant the supernatant and wash the pellet with 70% ethanol twice.
- Dry the pellet in a speed vac or over a clean piece of paper towel for 30 min.
- Resuspend the pellet in 50µL of ddH2O and measure the DNA concentration using Nanodrop.
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