1. Determining the total number of green fluorescent cells
The total number of colonies is 107.
2. Determining the amount of pGLO plasmid DNA in the bacterial cells spread on the LB/amp/ara plate.
2.
a. Determining the total amount of DNA
(DNA in µg) = (concentration of DNA in µg/µl) x (volume of DNA in µl)
= 0.08 µg/µl x 10 µl
= 0.8 µg
The total amount of pGLO DNA used in this experiment is 0.8 µg.
· What will this number tell you?
This piece of information will help us calculate the amount of DNA spread on the agar plate to determine the transformation efficiency.
This piece of information will help us calculate the amount of DNA spread on the agar plate to determine the transformation efficiency.
b. Determining the fraction of pGLO plasmid DNA (in the bacteria) that actually got spread onto the LB/amp/ara plate
Fraction of DNA used = Volume spread on a LB / amp plate (µl) / Total sample volume in text tube (µl)
= 100 µl / 510 µl
= 0.20
= 0.20
The fraction of pGLO plasmid DNA spread on the LB/amp/ara plate is 0.20.
· What will this number tell you?
This piece of information will also help us calculate the amount of DNA spread on the agar plate to determine the transformation efficiency.
This piece of information will also help us calculate the amount of DNA spread on the agar plate to determine the transformation efficiency.
So, how many micrograms of DNA did you spread on the LB/amp/ara plates?
pGLO DNA spread (µg) = Total amount of DNA used (µg) x fraction of DNA
= 0.8 µg x 0.20
= 0.16 µg
There are 0.16 micrograms of pGLO DNA was spread on the LB / amp / ara plates.
There are 0.16 micrograms of pGLO DNA was spread on the LB / amp / ara plates.
Number of colonies on the LB / amp / ara plate =
|
107
|
Micrograms of pGLO DNA spread on the plates
|
0.16 µg
|
Transformation efficiency = Total number of cells growing on the agar plate / Amount of DNA spread on the agar plate
= 107 / 0.16 µg
= 668.75
The transformation efficiency of this experiment is 669 tranformants/ µg.
Analysis
· Scientists would report 10,000 tranformants/ µg as 104 tranformants/ µg.
· Scientists would report 40,000 tranformants/ µg as 4 x 104 tranformants/ µg.
· Scientists would report 960,000 tranformants/ µg as 9.6 105 tranformants/ µg.
· Scientists would report 669 tranformants/ µg as 6.69 x 102 tranformants/ µg.
· Use a sentence or two to explain what your calculation of transformation efficiency means:
This number should indicate how effective we were in getting DNA molecules into bacterial cells. The transformation efficiency represents the total number of bacterial cells that expresses the green protein divided by the amount of DNA used in the experiment.
· How does your transformation efficiency compare with the above?
Compared to the values indicated by biotechnologists, our values are generally low. The transformation protocol has a transformation efficiency of between 8.0 x 102 and 7.0 x 103, whereas the number we calculated is 6.69 x 102.
· Calculate the transformation efficiency.
Number of colonies on LB/amp/ara plate
|
227 colonies
|
Micrograms of DNA spread on the plates
|
Total amount of pGLO DNA used in the experiment
= ( 10 µl ) x ( 0.08 µg/ µl)
= 0.8 µg
Fraction of DNA = ( 100 µl) / ( 250+10+250 µl)
= 0.20
pGLO DNA spread = (0.8 µg) x (0.20)
= 0.16 µg
|
Transformation efficiency =
|
= 227 colonies / 0.16 µg
= 1418.75 tranformants/ µg.
= 1.4 x 103 tranformants/ µg.
|
· Extra Credit Challenge
Transformation efficiency = 3 x 103 bacteria/ µg
Micrograms of DNA spread on the plates = 0.16 µg
Number of colonies = Transformation efficiency x Micrograms of DNA spread
= 3 x 103 bacteria/ µg x 0.16 µg
= 480 colonies
480 transformant colonies would be expected to grow on the LB/amp/ara plate.
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