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MBTA Project

DNA Extraction for MBTA Swabs

PowerSoil DNA Isolation Kit Protocol Modifications (for MBTA Samples) This protocol was written by Tiffany Hsu on 06/27/13. It was modified by Tiffany, Regina Joice, and Xochitl Morgan.

Notes:

  • The step numbers written here should correspond or be re-arranged from the step numbers in the Experienced User Protocol.
  • It may also be worth buying individual tubes and filling them with beads manually if this protocol becomes common.
  • They do sell vacuum manifolds for speeding up step 16, if this becomes a need.
  • We also made a habit of taking pictures of the swabs. The dirtier the swab, the more likely it was to give a universal 16S band.
  • Write the sample name on the top AND side of the glass bead tubes. Names will rub off the tops in the homogenizer!
  • Add 750 ul of Bead Solution.
  • Check Solution C1 to make sure it is not precipitated, then add 60 ul to each tube. If precipitated, head solution to 60°C until dissolved before use.
  • Snap off the swab head (while taking as little of the wooden part as possible) into the glass bead tube. Process only 1 swab per tube; otherwise, the swab will absorb too much solution. If doing multiple samples, use multiple glass bead tubes.
  • Invert the tubes or vortex to mix beads, swab heads, and solution.
  • Bead Beating Options: Homogenize at 6.0 m/s for 40 seconds.
  • Centrifuge bead tubes at 10,000 x g for 1 min at RT.
  • Transfer the supernatant to a clean 2 ml Collection Tube (provided). We should be able to recover about 400-500 ul supernatant. Do not worry about recovering some beads. You may need to poke around the swab or into the beads to get as much liquid as possible. These will be pelleted and removed in subsequent steps.
  • Add 250 ul of Solution C2 and vortex for 5 seconds. Incubate at 4°C for 5 min.
  • Centrifuge the tubes at room temperature for 1 min at 10,000 x g.
  • Avoiding the pellet, transfer up to 600 ul of supernatant to a clean 2 ml Collection Tube (provided).
  • Add 200 ul of Solution C3 and vortex briefly. Incubate @ 4°C for 5 min.
  • Centrifuge the tubes at RT for 1 min at 10,000 x g.
  • Avoiding the pellet, transfer up to 750 ul of supernatant into a clean 2 ml Collection Tube (provided).
  • Shake to mix Solution C4. Add 1200 ul to the supernatant and invert to mix. (The protocol says to vortex for 5 seconds, but it doesn’t seem to mix well).
  • Load 675 ul into a Spin Filter and centrifuge at 10,000 x g for 1 minute at RT. Discard the flow through and repeat 2x. At this point, you can combine your swabs. For example, if you had 2 swabs for a sample – you would load the filter 6x; if you had 3 swabs – 9x.
  • Add 500 ul of Solution C5 and centrifuge at room temperature for 30 seconds at 10,000 x g.
  • Discard the flow through.
  • Centrifuge again at RT for 1 min at 10,000 x g.
  • Carefully place spin filter in a clean 2 ml Collection Tube (provided). Avoid splashing any of Solution C5 onto the Spin Filter.
  • Add 100 ul of Solution C6 to the center of the white filter membrane. C6 DOES NOT contain EDTA.
  • Centrifuge at RT for 30 sec at 10,000 x g.
  • Aliquot 30 ul into 2 other tubes. This step can be changed; it is mostly for us to avoid freeze-thaws. Furthermore, the Broad requires at least 20 ul of sample.
  • Store samples at -20C.

Updated