I have had a couple of people ask me for bacterial DNA extraction protocols this semester, so I am going to publish them on this blog this week. One is an SDS thermal extraction for gram-negatives, and the second is a quick boil method for gram-positives. Feel free to copy and paste and make them your own!
Protocol#1
Spin down 500 µl of culture in a 1.5 mL tube at 12,000 rpm for 3 minutes. Repeat to increase size of base sample; total volume of sample spun down not to exceed 4.5 mL. Re-suspend cells in 0.3 mL TBS and 15 µL of 10% SDS (0.5% SDS final concentration). Incubate the solution in 55°C heat block for 10 minutes. Cool down to room temperature. Add 5 µl proteinase K solution @ dilution ratio of 5 mg powdered proteinase K per 250 µl of sterile molecular-biology grade water. Vortex ~20-30 seconds. Incubate the sample on ice for 5 minutes. Spin the sample at 12,000 rpm for 3 minutes. Verify the pellet is not loose. Transfer supernatant to a clean 1.5 mL tube. Add 300 µL of room temperature isopropanol. Gently mix by inversion until the chromosomal DNA threads to form a visible mass. Centrifuge the DNA at 12,000 rpm for 2 minutes. Carefully pour off the supernatant, use a clean pipette to remove any remaining supernatant, and add 300 µL room temperature 70% ethanol. Invert several times to wash the DNA pellet. Centrifuge at 12,000 rpm for 2 minutes. Drain off the supernatant, use a clean pipette to remove any remaining supernatant, and allow the DNA pellet to air dry under hood for 15-20 minutes. Re-suspend the DNA pellet in 50 µL sterile molecular-biology grade water.
Protocol #2:
Incubate culture for 24 hours @ 37°C. Pellet 500 µl of culture at 12,000 x g for 3 minutes. Re -suspend pellet in 500 µl sterile water using a 30 second vortex. Incubate sample for 10 minutes @ 100°C. Pellet @ 12,000 g for 3 minutes. Pour off supernatant into 1.5 ml Eppe tube and discard pellet.
Until next week, please enjoy this National Geographic article about Homo naledi, a human ancestor recently discovered in a South African cave (artist rendition below):
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