Glowing Bacteria:
Transformation Efficiency
Purpose: To determine how well your E. coli cells
took up and expressed GFP after transformation.
Background:
Transformation efficiency is a quantitative value that describes how
effective you were at getting plasmid DNA into your E. coli bacteria. The
number represents how many cells were transformed per microgram (µg) of
plasmid DNA used. This calculation requires two values: the number of cells
that were successfully transformed and the amount of plasmid DNA used for
the transformation.
Procedures:
1. Each colony on your plates can be assumed to be derived from a single
cell, so the most direct way to determine the total number of successfully
transformed cells is to count the colonies on the LB/Amp/Arabinose plate.
Enter that number here: ________ colonies
2. The total amount (µg) of pGLO plasmid DNA that was in your +pGLO
tube is equal to the product of the concentration and the volume used:
Amount DNA (µg) = Concentration of DNA (µg/µL) x Volume of DNA (µL)
You used 10µL of plasmid solution with a concentration of 0.08µg/µL:
0.08 (µg/µL) x 10 (µL) = ________ µg total plasmid DNA
3. Since the amount of DNA calculated above was used for multiple plates,
you have to calculate the fraction of this amount that was actually spread
onto the LB/Amp/Arabinose plate.
Fraction of DNA used = Volume spread on LB/Amp/Arabinose plate
Total volume in tube
You spread 100µL of solution from a tube containing 510µL total volume:
100µL ÷ 510µL = ______ (Fraction of DNA spread on plate)
4. To determine the actual amount of DNA spread on the plate, multiply the
total amount of DNA used (#2) by the fraction of DNA spread on the
LB/Amp/Arabinose plate (#3):
(#2)______ µg total plasmid DNA x (#3)______ (Fraction of DNA spread on plate)
= ______ µg plasmid DNA spread on plate
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5. Transformation efficiency is equal to the number of cells that were
transformed (#1) per amount (µg) plasmid DNA spread on the plate (#4):
(#1) ______ colonies on plate
÷
(#4) _____ µg plasmid DNA spread on plate
= _________ transformants per µg plasmid DNA
(Transformation efficiency)
Analysis Questions:
1. Explain what your calculation of transformation efficiency means.
2. The transformation protocol you used generally has a transformation
efficiency of 800-7000 transformants per µg plasmid DNA. How does
your calculation compare with this?
3. Report the transformation efficiency of several groups from your class.
Group
Transformation Efficiency
4. How does your transformation efficiency compare with theirs?
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