Shlo/directmagneticprotocol

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Written by Stephanie Lo, for Harvard iGEM 2007 use only ...
Direct Magnetic Bead Assay

Protocol

  1. Use 20 - 25 ul of beads; in our case, TALON beads for his-tagged proteins and streptavidin beads for Strep2-tagged proteins.
    1. Location: Refrigerator near 37C plate incubator in small room
    2. Be sure to vortex before using beads; they tend to settle.
  2. Use 1000 ul of cells, at OD of beginning to mid log. If you're planning to mix cultures with different resistances, remember to pellet, remove supernatant, and resuspend in a friendly medium first
  3. In 1.5mL microcentrifuge tube, combine cells and beads. Pipet up and down gently to ensure mixing.
  4. For 10-30 minutes, put tubes on refrigerated rotissieres.
  5. Afterward, use bead rack to pull beads to side. Allow at least 5 minutes for all beads to pull to side.
  6. Leaving the tubes in the rack, use a yellow pipet (blues are too big) to suck up supernatant. Be careful not to disturb the beads on the side.
    1. The supernatant can be disposed of, but it is interesting/nice to keep the supernatant from the first rotissiere to measure how many cells were removed. A negative control, therefore, should have a high supernatant OD.
  7. Remove the tubes from the rack and resuspend in 1000 ul washing solution (imidazole, PBS, MACS buffer). Again, pipet up and down to ensure that the beads are evenly resuspended, and ensure that there is no pellet on the side of the tube.
  8. Repeat rotissiere, etc. until 3-5 washes have been completed.
  9. After last wash, resuspend in 100-500ul LB with antibiotic, as appropriate.
  10. Plate cells. The amount will depend on your starting OD and final dilution. If you have a starting OD of about 0.4 and did 3 washes, and reconstituted in 500 ul, a 50ul plate should have a good number of colonies (though probably on the high side already).



Notes

Strep beads are less sticky than TALON beads, so higher OD may be necessary to get good results. I've gotten good numbers with starting OD of 0.390, using 700ul of each (sender/reciever: so 1400ul total cells), 3 washes, and reconstitution of 750ul final before a plating of 75ul. Effectively, this is plating an OD around 0.0546 (not accounting for loss of cells throughout wash).

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