Rapid Method for Preparation of Genomic DNA from P. aeruginosa
- Inoculate 1 ml of L-broth or other rich media with cells of the strain from which genomic DNA is to be isolated. Grow this culture at 37°C overnight on a roller.
- Transfer this overnight culture to a 1.7 ml microfuge tube. Pellet the cells by centrifugation on high at room temperature for one minute.
- Resuspend and wash the cells in 1 ml of TNE buffer by pipetting gently up and down until the pellet is completely resuspended.
- Pellet the cells by centrifuging as before.
- Resuspend the pellet in 135 µl TNE buffer.
- Add 135 µl of TNE buffer containing 2% Triton X-100.
- Add 30 µl freshly prepared lysozyme (5 mg/ml). Mix well by tapping the tube.
- Incubate in a 37°C water bath for 30 minutes.
- Add 15 µl proteinase K solution (20 mg/ml). Mix well by inverting the tube several times.
- Incubate in a 65°C waterbath for 2 hours.
- Genomic DNA thus prepared may be kept at -20°C until used.
- Digestion of the DNA is best accomplished by allowing the digestion to proceed overnight, and by keeping the volume of DNA to less than one-fifth the total volume of the digestion mixture. There is sufficient DNA in each 50 µl digestion for three electrophoretic runs on a 14.5 x 20 cm gel suitable for Southern blotting.
DNA preparation 8.4 µl TNE Buffer dH2O 34.6 µl 10X enzyme buffer 5.0 µl Tris-HCl, pH 8.0 10 mM Restriction enzyme (20-30 U) 2.0 µl NaCl 10 mM Total volume: 50.0 µl EDTA, pH 8.0 10 mM
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