Field of Science

Do competent cells stick to an agar layer containing DNA?

Apparently not.

I made plates of agar culture medium topped with a thin layer of agar containing chromosomal DNA (1 µg/ml or 10 µg/ml).  I layered ~ 10^8 cells (log phase or competent) on top of the DNA-agar, let them sit for 10 min, and then washed the surface by flowing buffer over it (2 x 25 ml).  Then I overlaid the agar with 1 ml  of medium ± DNase I.  Finally I washed the agar surface one last time with 25 ml of buffer and plated both this final wash and the top agar itself. 

If the competent cells were able to bind to the DNA I had mixed into the top agar, I expected to see many more competent cells than log-phase cells in the top agar when DNase I wasn't used.  And when DNase I was used, I expected Wash 3 to contain most of the cells that would otherwise be with the top agar.  But neither of these effects were seen.

I don't know if the differences between the log-phase cells and the competent cells (leftmost bars vs all the other bars) are significant.  It could just be because the log cells were my first attempt.

But there are clearly no significant differences caused by treatment with DNase I, nor by use of the two different DNA concentrations.

Controls I failed to do: 
  • Top agar with no DNA.  This would control for non-specific sticking.
  • Transformation of the cells by the NovR allele in the DNA.  If this gives no transformants, maybe none of the DNA was exposed above the agar surface.  If it gives lots of transformants, maybe the exposed DNA was all released from the agar into the medium.
Other changes I could try:  There's no reason to put the DNA-agar on top of normal sBHI agar, since I'm not going to try to grow the cells.  Instead I can just make thin layers of agarose with DNA, varying both the agarose concentration and the DNA concentration.  I could try higher DNA concentrations, up to 100 µg/ml.  For ease of washing I could make these on glass slides rather than in Petri dishes.  (Nope - just tried that and the agar just slid off the slide when I washed water over it.)

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