Team:Calgary/Notebook/Protocols/Process24
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<li>100% ethanol</li> | <li>100% ethanol</li> | ||
<li>70% ethanol</li> | <li>70% ethanol</li> | ||
- | <li> | + | <li>ddH<sub>2</sub>O</li> |
<p><h4>Protocol</h4></p> | <p><h4>Protocol</h4></p> | ||
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<li>Decant the supernatant and wash the pellet with 70% ethanol twice.</li> | <li>Decant the supernatant and wash the pellet with 70% ethanol twice.</li> | ||
<li>Dry the pellet in a speed vac or over a clean piece of paper towel for 30 min.</li> | <li>Dry the pellet in a speed vac or over a clean piece of paper towel for 30 min.</li> | ||
- | <li>Resuspend the pellet in 50µL of | + | <li>Resuspend the pellet in 50µL of ddH<sub>2</sub>O and measure the DNA concentration using Nanodrop.</li> |
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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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