1. Lecture 9
January 23, 2016
Biotech 3

2. 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)

3. 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

4. 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

5. 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

6. 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