Field of Science

Do cells bind to DNA stuck on beads?

I need to plan the experiment(s) where I test whether competent cells bind to DNA on magnetic beads.

I have 1 ml each of two types of 1µ and 2.8µ magnetic 'dynabeads'.  The beads have been coated with streptavidin, and one batch of each size has then been blocked with BSA, which apparently reduces the surface charge and makes them better for binding protein but not so good for nucleic acids and I think more prone to clumping in high-salt buffers.  I don't know how the different surfaces will affect non-specific cell binding, something I want to avoid (Invitrogen recommends  including 0.01-0.1% Tween 20 to reduce non-specific binding, where that won't interfere with the assay).  And I don't know how the surface properties of these  beads compare to the properties of the polystyrene beads I'll be using for the tweezers experiments.  (I need to keep reminding myself that I'm only using the magnetic beads to check whether cells will bind to DNA on beads, so I shouldn't waste a lot of time optimizing the assay.)

I can separate the beads from 150 µl of liquid by simply drawing the mixture up in a pipette tip, holding the tip in the nanobead magnet rack for 10 seconds, and slowly expelling the liquid; almost all the beads remain behind on the side of the tip.  I can then resuspend the beads by drawing clean buffer up into the tip, away from the magnet.

So:  First mix one aliquot of beads with DNA (Invitrogen says to do this in TE + 1 M NaCl).  To start I'll just use one size of beads, arbitrarily the 1 µ ones.  I know that the stocks contain 10 mg beads per ml, but I don't know how many beads this is.  I was going to find that out, by diluting some beads and looking at them under the microscope (we have a hemocytometer).  But here's a rough calculation:  If the density of the beads is a bit higher than that of water, then a single bead 1 µ in diameter will have the same mass as a 1 µ cube of water, which is 10^-9 mg.  So 10 mg of beads/ml is ~10^10 beads/ml.  As I did with the polystyrene beads, I'll wash the beads several times to remove unbound DNA.  I don't think I need to check that DNA has bound by using the sensitive fluorescence assay - I'll leave that to do if I don't see a difference in cells associated with beads with and without DNA.

Then I'll thaw some frozen competent H. influenzae cells, wash away the glycerol and resuspend them in BHI.  Then I'll  mix them with DNA-treated beads and control (no DNA) beads, at a concentration of, say,  ~ 5x10^8 cells and beads per ml.  I'll incubate cells plus beads briefly (1 min at 37°C?) and then wash the beads three times, saving the eluate/supernatant/whatever it should be called.  Then I'll plate the beads (several dilutions) and the eluates.  I'll also look at the beads under the microscope (maybe do that first).
  1. Make washing buffer, wash beads, resuspend in BHI, in MIV and in PBS.  
  2. Check under microscope to see if they're clumping.
  3. Incubate beads with DNA, wash well.Thaw and wash cells.  Resuspend in BHI or MIV.
  4. Mix cells with beads, incubate briefly and wash.
  5. Assess binding by plating and/or microscopy.

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