A640 nm of Hfr Cells Number of Hfr (Cfu/ml) from standard curve Number of Hfr (C

Question

A640 nm of Hfr Cells Number of Hfr (Cfu/ml) from standard curve Number of Hfr (Cfu/ml) in reaction 8. Xo (Cfu/ml from graph x 2[dilution factor Growth of MMD plus streptomycin minus leucine (+/-) Growth on LB plates (+1-) Organisnm F- Hfr Colonies per plate Cfu/ml F- and Hfr Mix 10' 102 10 101 MMD is minimal medium supplemented with amino acids necessary for growth of F. The indicated amino acid leucine UTC has been left out of the medium. Average number of recombinant cells (Cfu/ml) Percent recombination

Explanation / Answer

The hfr and F- cannot grow on the selective plates, when cultured separately. The Hfr cell number is 1.6 * 10^8 / ml.
The F- and Hfr mix has colonies 220 and 24 on 10^4 and 10^3 dilutions respectively. The formula to find out the CFU/ml from colonies on plate is
CFU /ml = colonies counted * dilution factor / volume plated
colonies = 220; dilution factor = 10^3; volume is 0.1ml ( this volume is what we spread on plates 100ul).
CFU/ml = 220 * 10^3 / 0.1 = 2.20 * 10^6 /ml

CFU / ml = 24 * 10^4 / 0.1 = 2.4 * 10^6 /ml

Average CFU / ml = (2.20 * 10^6 + 2.4 * 10^6) / 2 = 2.3* 10^6 cfu /ml

The percent recombination = number of recombinants / total number of cells
= 2.3* 10^6 / 1.6 * 10^8
= 1.43%.

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