Transcript Glowing Bacteria! - Mrs. Basepayne's Science Spot

Purpose:
To Transform E. coli bacteria with
the jellyfish gene GFP
Procedures
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Set up an ice water bath.
Label one microfuge tube containing 150 ul of CaCl2:
“+pGFP DNA”
Label the second microfuge tube containing 150 ul of
CaCl2: “- control”
Place both tubes in your ice bath
From a starter plate using an inoculating loop
transfer about ¼ loop-full of E. coli from the plate to
each tube. Do NOT transfer any agar and twirl the
loop too release the bacteria.
Procedures continued
6. The CaCl2 tubes should be cloudy with E. coli now. Mix
the bacteria thoroughly by gently pipetting up and down
4-5 times. The mixture should be cloudy. If it is not, add
more bacteria.
7. Place both tubes with the CaCl2/E. coli mix back on ice.
8. Using a fresh tip transfer 10 ul of pGFP plasmid DNA to
the tube labeled +pGFP DNA ONLY! Mix this tube by
pipetting up and down 5 – 10 times. Gently tap the tube
to make sure the mixture settles back on the bottom of
the tube.

DO NOT ADD PLASMID DNA TO YOUR NEGATIVE
CONTROL!
9. Place both tubs on ice and incubate on ice for 15 minutes.
Procedures Continued
10. While the tubes are incubating on ice obtain two luria +ampicillin
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plates. Label the bottom of one plate with your initials, the date,
and “+GFP DNA”. Label the bottom of a second plate with your
initials, the date, and “- control”.
After 15 minutes perform a “heat shock”. Instantly remove both
tubes form the ice and incubate them in the 42°C water bath for 60
seconds OR for 3 minutes in you hands.
Add 200 ul of LB to recover your bacteria.
Transfer 100 ul of your “- control” tube to the negative control
plate. Use a bacti-spreader to spread the liquid across the entire
plate.
Using a fresh tip transfer 100 ul of your “+ pGFP DNA” tube to the
“+ pGFP DNA” plate. Use a fresh bacti-spreader to spread the
liquid across the entire plate.
Let cells penetrate the agar for 3 minutes.
Incubate upside down overnight at 37°C in the incubator.
Observe the plates under a UV light. Count and record the
number of fluorescent colonies on each plate. Record your results.
Analysis
 Calculate the transformation efficiency:
 Total mass of the plasmid (total mass = volume x
concentration)
 Total volume of the suspension
 Fraction of cell suspension on plate (ul on plate/total
volume)
 Total mass of plasmid in fraction (mass of plasmid x
fraction on the plate)
 Number of colonies per ug of plasmid (# of colonies
counted/mass of plasmid on the plate)
Assessment Questions
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Define bacterial transformation.
What is E. coli such a convenient host for
transformation in biotechnology?
Define a vector. What type of vector is used in this
protocol?
In this laboratory, how did you select for culturing
only transformed cells? What happened to the cells
you failed to transform?
Define the term “reporter gene”. What reporter gene
was used in this laboratory?
Assessment Questions
6. What is a microbial colony? What does one colongy
represent on your experimental (+pGFP) Luria
ampicillin agar plate?
7. To make E. coli “competent” at being transformed,
the cells are re-suspended in CaCl2 solution. What is
the purpose of the CaCl2?
8. List some reasons why scientists transform E coli
with plasmids?