Bacterial Transformation Lab
Lab Objective: To understand that the genetic composition of cells can be altered by incorporation
of exogenous DNA into cells to form novel (new) combinations forming GMO genetically modified
organisms that are also transgenic.
Define new terms:
o
o
o
o
exogenous DNA
transformation
GMO
Transgenic organisms
Question: (rephrase objective)
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Hypothesis: (Testable Prediction)
If _____________________________________________________________then
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List Materials (with labeled drawings):
1. What is the purpose of using ampicillin on the plates? ___________________________________
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2. What genes are present in the plasmid and what is the function of each gene?
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Label and explain a possible plasmid for this lab. (Hint: R plasmids are used)
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CELLS Solution is prepared day(s)
prior to the lab.
1. Using a sterile inoculating loop,
several colonies of bacteria (each
“dot” is a colony) are ‘picked’
from the top of the LB agar class
starter plate.
2. Transfer the colonies to tubes of
cold Ca++Cl solution.
3. Swirl and twist the loop/tip to
make sure all the bacteria mixed
in the solution.
* Why place the bacteria cells in a Ca++Cl solution?
4. Freeze
Drawings
Lab Day Protocol
1.
DRAW these tubes. Explain why we need 2 tubes?
Label two microtubes:
one with a (+)
one with a (-)
2.
DRAW the 3 Plates:
Plate 1: LB (food)
Plate 2: LB / Amp (ampicillin)
Mark a LINE down the middle of plates
1 and 2 (NOT plate 3).
On one half place a
+
On the other half place a
-
Plate 3: LB / Amp / arabinose
Mark a + on this plate.
3.
Using a micropipette set at 050:
Add 50 μL of CELLS solution to
both the + and – tubes
(E.coli bacteria cells)
DRAW this step:
4.
Using a second (smaller volume)
micropipette set at 10.0:
Use a sterile pipette tips, add 10 μL of
rpARA plasmid to the positive (+) tube
only.
Tap gently to mix.
Optional:
Add 10 μL steril water to the (-) tube.
Tap gently to mix.
5.
Incubate both + and - tubes on ice for
10 minutes.
Start Time: _________
6.
Heat shock: Take your tubes in the ice
water cup to the 42°C hot water bath.
Why HEAT shock the tubes?
Make sure the water bath is at 42°C.
Transfer the tubes from ice to the hot
water for 45 - 50 seconds Heat Shock.
Immediately return the tubes to the ice
for 2 minutes.
7.
Place + and - tubes in rack at room
temperature.
DRAW
Normalize bacteria cells:
Add 150 μL LB broth to both tubes.
MIX / vortex gently.
Rest tubes for 2 minutes.
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Plating Petri Dishes
8.
Set Micropipette at 050.
Use steril tip for – loading. Use a
new steril tip for + loading.
DRAW setting
Why ICE now?
-- Tube:
50 μL onto Plates I and 2. Be sure
to place on – side.
DRAW
+ Tube:
50 μL onto Plates I and 2. Be sure
to place on + side.
AND
50 μL onto Plates 3.
9.
Using a steril inoculation loop, gently
spread the – sides of plates 1 and 2.
DRAW
Using a steril inoculation loop, gently
spread the + sides of plates 1 and 2,
and then spread plate 3.
10. Cover each plate and stack.
Why incubate at 37o for 24 hours?
After 15 minutes invert plates (agar side
up).
Tape & Label with team names.
Incubate at 37o for 24 hours.
Data / Results:
1. What do all of the cells growing on the LB/amp and LB/amp/ara plates have in common?
2. Explain the controls used in this experiment.
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3. Explain the TRANSFORMATION that has occurred. (Be specific and use Central Dogma
concepts)
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Reflection / Extension
1. What are the benefits of this technology to society? Give specific examples.
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2. Identify and discuss any ethical issue(s) that may be raised by the technology used in this lab.
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