Use the following web server and the data above to determine the transformation efficiency of pUC18 in units of transformants per ug DNA.
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Supreeta N.
Transformation efficiency is expressed as the number of antibiotic-resistant colonies per ug of plasmid DNA. The object is to determine the mass of plasmid that was spread on the experimental plate and that was, therefore, responsible for the transformants (the number of colonies) observed. Because transformation is limited to only those cells that are competent, increasing the amount of plasmid used does not necessarily increase the probability that a cell will be transformed. A sample of competent cells is usually saturated with the addition of a small amount of plasmid, and excess DNA may actually interfere with the transformation process. a. Determine the total mass (in ug) of plasmid used. Remember, you used 10 μL of plasmid at a concentration of 0.005 ug/αL. b. Calculate the total volume of cell suspension prepared. c. Now calculate the fraction of the total cell suspension that was spread on the plate. d. Determine the mass of plasmid in the cell suspension spread. e. Determine the number of colonies per μg plasmid DNA. Express your answer in scientific notation.
Josee P.
Exercise C: Calculating transformation efficiency Table 1. Transformation efficiency of E. coli cells transformed with plasmid pUC19 Plate | Plasmid DNA added? Y or N | DNA Diluted? | Volume of DNA used (µl) | Concentration of DNA (ng/µl) | Total DNA used in transformation (ng)* | # transformants (# of colonies) | Transformation efficiency ---|---|---|---|---|---|---|--- LBA - | | | | n/a | | n/a, LAWN | n/a LBA + | | | 10 | 10 ng/µl | | n/a, LAWN | n/a LBA/amp - | | | | n/a | | | LBA/amp + | | 1X (not diluted) | 10 | 20 ng/µl | | | LBA/amp + | | 0.5X (diluted by 1/2) | 10 | 10 ng/µl | | | *To calculate "total DNA used in transformation," multiple column 3 and 4 together (the volume units should cancel out).
Adi S.
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