aureus genomic DNA isolation-PDF

Overview

Column-based methods for DNA purification were optimized for E. coli and may not give good yields for other types of bacteria. The following is a method for isolating the genomic DNA of S. aureus, but could easily be adapted for the isolation of other gram-positive bacteria. The protocol consists of cell lysis, degradation of the heat-stable nuclease using proteinase K, phenol-chloroform extraction, and ethanol precipitation.

Materials

• 1.5 mL microcentrifuge tubes
• Lysostaphin solution (200 ug/mL lysostaphin; 20 mM Tris-HCl, pH 7.5; 10 mM EDTA, pH 8.0)
• Proteinase K
• Phenol:Chloroform: Isoamyl Alcohol (25:24:1)
• Tris-EDTA buffer (TE) (10 mM Tris-HCl, pH 7.5; 1 mM EDTA, pH 8.0)
• Sodium chloride solution (other salts, such as sodium acetate, may also be used)
• Ethanol (100% and 70%)

Procedure

1. Pipet 1 mL of overnight bacterial culture into a 1.5 mL microcentrifuge tube, and centrifuge for 2 minutes at 5000 g to pellet the bacteria.
   (Note: using culture volumes greater than 1 mL may lead to solutions that are too viscous to easily pipet.)
2. Discard the supernatant and re-suspend the bacteria in 500 μL of lysostaphin solution (200 μg/mL lysostaphin; 20 mM Tris-HCl, pH 7.5; 10 mM EDTA).
3. Incubate at 37 °C for at least thirty minutes. The culture does not need to be completely clear at the end of this step.
4. Add proteinase K to a final concentration of 100 μg/mL and incubate at 56 °C for an hour.
5. Add 25:24:1 buffered phenol:chloroform: isoamyl alcohol to the culture at a ratio of 1:1. Mix thoroughly by inversion, but don’t vortex.
6. Centrifuge at max speed (~15,000 – 17,000 x g) for 10 minutes.
7. Pipet off the aqueous top layer, being careful not to disturb the white layer at the interface. Transfer the top layer to a new microcentrifuge tube, and save the old tube (you can go back to it if necessary).
8. Repeat steps 5-7 until the layer of debris under the aqueous top layer is small. After the first centrifugation, you can reduce the time of the spin to five minutes.
9. To extract any remaining phenol, add chloroform at a ratio of 1:1, mix by inversion, and centrifuge at max speed for 5 minutes. Add TE buffer to the solution if the volume is getting too low to handle cleanly.
10. Pipet the top layer into a new 1.5 mL microcentrifuge tube. Make sure that there is enough room in the tube to add salt and ethanol (e.g. 350 μL DNA, ~128 μL 2 M NaCl, and 800 μL EtOH).
11. Add NaCl to a final concentration of 200 mM (taking into account the ethanol added in the next step) and mix by flicking gently.
12. Add two volumes of 100% ethanol and mix by inversion.
    (Optional: put tube in the -20 °C freezer for 30 min. to overnight to increase yield.)
13. Centrifuge at max speed for 10 minutes to pellet the DNA.
14. Pipet the supernatant into a separate microcentrifuge tube, being careful not to dislodge the pellet. If the pellet does become dislodged, repeat the centrifugation.
    (Optional: to increase DNA yield, put the supernatant in the -20 °C freezer overnight, then pellet the DNA and continue with the protocol from this step.)
15. Add 1 mL of 70% ethanol to the pellet. Roll the tube to make sure that the ethanol washes on all sides of the tube, the lid, and the pellet. It is okay if the pellet becomes dislodged at this stage.
16. Centrifuge for a few seconds to remove ethanol from the lid (longer if the pellet does become dislodged).
17. Pipet out all of the liquid, and discard. Let the pellet dry for a few minutes.
18. Again, centrifuge for a few seconds and pipet all liquid out of the tube. Allow the pellet to dry until the edges start becoming transparent (~5 minutes or less).
    (Note: avoid over-drying the pellet. Once it turns glassy, it will be difficult to get back into solution. If the pellet does become too dry, try letting it soak in the 5 °C fridge overnight. If that doesn’t work, try heating it to 70-80 °C in a heat block.)
19. Add 100 μL TE directly to the pellet, making sure not to scratch the pellet so that DNA does not stick to the pipet tip.

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!