Thursday, May 7, 2009

Third transformation's a charm?

My third competent cell preparation and transformation looks like it was fairly successful. Not only were there transformants, but the numbers look a lot more reasonable.

Picking dilutions with reasonable numbers of colonies on the plates:

K = KanR / CFU = 1.54 e -2
N = NovR / CFU = 1.82 e -3
D = KanR NovR / CFU = 1.95 e -4

K*N = 2.8 e -5

D / (K*N) = 7.71

So there’s an excess of nearly 8x double transformants relative to expected. Since the Kan and Nov markers are linked, I can’t accurately measure either linkage or the fraction of the competent cells in the prep. This requires a third marker. I’ll next turn to my frozen stocks and use a third antibiotic resistance marker that’s unlinked from KanR and NovR, perhaps SpcR (spectinomycin resistance). This should allow me to measure the fraction of competent cells in the culture and use this to correct the apparent linkage of Kan and Nov to an accurate measurement.

Notably, the KanR and NovR rates were again about an order of magnitude different. So indeed, there’s substantial variation in transformation rates for different markers. The underlying cause of this variation is something I hope to get at with my planned genome-wide transformation rate measurements in the future.

Other issues:
  • The culture’s cell density still looks a bit high (1.4 e 9 / ml), but it’s <1/3>
  • My NO DNA CONTROLS also had a few colonies on the Kan plates, indicating either contaminants or a relatively high mutation rate of KanS -> KanR in Rd. I’ll check this by taking the KanR colonies from my negative control plates and streaking to LB to see if they are really Haemophilus. KanR / CFU = 1.8 e -5. This was still substantially lower than cells transformed with DNA, so the rates are still reasonably accurate.
  • Some of my plates (particularly the double antibiotic plates) had pretty uneven spreading and heterogeneous colony sizes. I suspect this was due to unevenly spreading fresh hemin onto the plates prior to spread the cell dilutions. I hadn’t yet carefully observed Sunita’s masterful plate spreading technique when I spread the hemin, but did by the time I spread the cells.

Time to make fresh media!

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