1. Name: Date:
PGLO Lab Practice
Cut out the descriptions of each step on the back page and match them to the pictures on the right.
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3. Please cut out the steps below and match them with the pictures on the previous page.
A. Label one mico test tube +pGLO and the other – B. Use a new sterile inoculating loop to spread the liquid
pGLO. The +pGLO is the one with the plasmids, the – evenly across the surface of the agar in a zig-zag pattern.
pGLO is the control without plasmids, just to compare. This spreads the bacteria out so it can reach all the
nutrients.
C. Heat shock. Using the foam rack as a holder, move D. Use a sterile inoculating loop to pick up a single spot
the microtubes into the water bath at 42°C for 50 of bacteria from the Petri dish. Put the loop in the
seconds. This causes the phospholipids that make up the microtubes and spin/twist the loop until all the bacteria
cell membrane of the bacteria to expand, allowing the is mixed into the solution.
plasmids to enter the bacteria.
E. Place the microtubes on ice. This brings down the F. Using a sterile pipette, add 250 microliters of LB
temperature to shrink the cell membrane of the bacteria, Broth to each of the microtubes. This gives the bacteria
making it more stretchy during heat shock. extra nutrients.
G. While your tubes are sitting on ice, label your four H. Using an new sterile pipette, add 100 microliters of
Petri dishes as +pLGO LB/amp, +pGLO LB/amp/ara, the transformation and control suspensions onto the
-pGLO LB/amp, and –pGLO LB. Label them on the appropriate Petri dishes.
bottom, not the lid, so if you switch the lids accidentally,
you still know which is which.
I. Put a new sterile inoculating loop into the plasmid J. Put the microtubes in a test tube rack, and then on ice
DNA microtube. There should be a layer of plasmid again, for 10 minutes. This brings down the
solution across the ring. Mix it into the +pGLO temperature to shrink the cell membrane of the bacteria,
microtube, but NOT the –pGLO microtube. We do this making it more stretchy during heat shock.
because the –pGLO is our control, to compare and make
sure we are growing the bacteria right.
K. Using a sterile pipette, transfer 250 microliters of L. Stack up your Petri dishes and tape them. Put them in
transformation solution (CaCl2) to the microtubes. This an incubator at 37°C for 24 hours. This warm
gives the plasmid DNA a negative charge, which allows temperature is the best environment for the E. coli to
it to pass through the cell membrane of the bactiera. grow in.