Lecture 9 January 23, 2016 Biotech 3

Part I – Module II: Calculate the transformation efficiency of the pUC8 ligated vector Transformation Efficiency – calculated by quantitating the number of bacteria (CFU) that were successfully transformed per microgram (μg) of DNA. Reported as CFU/ μg. Dependent on many factors: Type of bacteria Bacterial growth phase Amount of plasmid used DNA tertiary structure (supercoiled) Heat shock temperature Method used to transform cells Example: 108 vs 105 CFU/ug (electroporation vs chemically competent cells)

Calculate Transformation Efficiency Required information: Number of CFU Volume of bacterial culture plated Fraction of overall final transformation volume (i.e. dilution) Mass of the DNA used in the transformation Example: 50 ng of plasmid DNA is transformed into a final transformation volume of 500 μl, and 10 μl of this volume is spread onto a plate. Assume 60 CFU are observed on the plate. 1. Count colonies to determine CFU CFU = 60 DNA spread on the plate Volume spread (μl) X DNA transformation (μg) = 2. Determine amount of plasmid DNA (in μg) spread on the plate. Total volume of transformation (μl) 10 μl X 0.05 μg = = 0.001 μl 500 μl 3. Divide the number of colonies on the plate (in CFU) by the amount of DNA (in μg) spread on the plate. 60 CFU = 60 X104 CFU/μg 0.001 μg

Part II – Blue/White Cloning of DNA Fragment and Assay of β–galactosidase Module III B-galactosidase (Yellow) λmax = 420 nm Lac+: pUC8 without insert  β-galactosidase is expressed  blue colonies  yellow observed lac-: pUC8 with insert  β-galactosidase is not expressed  white colonies  no yellow observed

Part II – Blue/White Cloning of DNA Fragment and Assay of β–galactosidase Module III calculation Background: 1972, Jeffrey Miller published "Experiments in Molecular Genetics" which contained a protocol for determining the amount of β-Gal with ONPG. Because of this, ONPG/β-Gal assays are referred to as "Miller" assays, and a standardized amount of β-Gal activity is a "Miller Unit“. A420 – (1.75 x A550)) 1 Miller Unit = 1000 X T x V x A600 A420 is the absorbance of the yellow ONP A550 is the scatter from cell debris, which when multiplied by 1.75 approximates the scatter observed at A420 T = reaction time in minutes V = volume of culture assayed A600 reflects cell density

Part II – Blue/White Cloning of DNA Fragment and Assay of β–galactosidase Module III data collection Sample Time Real Time O.D. 420 O.D. 550 O.D. 600 Miller Units Lac+ T0 Lac- T0 Lac+ T1 30 min Lac- T1 Lac+ T2 90 min Lac- T2 Lac+ T3 120 min Lac- T3