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Watch this video about the pGLO Laboratory before coming to class. It previews and materials
and procedures in order to prepare you for the activity.
It may help to have the laboratory printout nearby.
Step 1: Read through procedure completely and gather all materials.
Here is an image of all the materials you will be provided. You can see a small container
with ice, a stopwatch, a marking pen, and a small biohazard waste container.
In addition, you will be provided 2 microcentrifuge tubes containing 100uL of competent E.coli.
These two tubes should be kept on ice. A third microcentrifuge tube contains 5uL of pGLO,
and you will have one or two additional microcentrifuge tubes containing SOC nutrient broth.
You will be using a p1000 and a p10 plus the tips that fit on those two pipetmen.
Included in the materials will be 4 poured agar plates. They contain different chemicals
- be sure to pay attention in class when your instructor reviews the differences.
These are inoculation loops, this is one version of a UV light pen, and you will need a foam
floater - it’s just like an inner tube for microcentrifuge tubes that keeps them afloat
in water.
With the rest of the class, you will share a few items: the 42 degree Celsius water bath,
the 37 degree Celsius water bath, a large Biohazard waste bin, and a 37 degree celsius
air incubator .
Step 2: Keep competent E.coli on ice as much as possible.
Step 3: Label on microcentrifuge tube containing E.coli +pGLO or positive
and the other microcentrifuge tube -pGLO or negative.
Step 4: Add 5uL of pGLO plasmid to the +pGLO tube and ONLY the +pGLO tube.
Set your p10 pipetteman to 5uL, put a tip on it, and remove 5uL of plasmid from the
tube. Remember to only go to the first stop. Put those 5uL in the +pGLO tube and place
it
back
on ice.