1. Protein Transformation Prep
• The Making of a Plasmid
• Equipment Id.
• Try Techniques
• Get supplies
• Flow chart
• Form groups
• Quiz

2.       Make two Plasmids gene Pest Resistance Factor VIII
Glue/Tape
Tuck Under & Glue/Tape
Glue/Tape
Tuck Under & Glue/Tape
Pick a Fluorescent Protein gene to insert
mCherry
mTangerine
mBanana
GFP
Green Fluorescent Protein
BFP
Blue Fluorescent Protein
mGrape
mPlum 
Restriction Enzyme
Use DNA Ligase (tape or glue) to bond the gene of interest
Restriction Enzyme
Restriction Enzyme
Add an antibiotic resistance gene to both plasmids you make.  
AmpR gene
Ampicillin resistance
TetraR
gene
Tetracycline resistance
KanR gene
Kanamycin resistance
PenR gene
Penicillin resistance
Time to make the second Plasmid Model 
Restriction Enzyme 
Restriction Enzyme
Use DNA Ligase (tape or glue) to bond the gene of interest
Congratulations scientists you have just made recombinant DNA: genetically engineered DNA with genes that can save lives! 
Pick a gene of interest to add to the second plasmid you make
Restriction Enzyme
Pest Resistance
gene
Save a plant
Insulin gene
Save a diabetic
Factor VIII
gene
Save a hemophiliac
Oil Spill gene
Save an environment

3. Make a Plasmid Activity
1. Cut the DNA with a _______________ (Scissors)
2. My gene of interest was (FP - ________ & __________)
3. My goal is to (FP) - track ____________; save ________
4. The petri dish would have:
___________ antibiotic; ___________ antibiotic
so…I need to make the transformed bacteria
resistant to that antibiotic (_____); (_____)
5. What I have made are 2 small circular pieces of DNA
with two genes of interest each & they called plasmids.

4. 4 microcentrifuge tubes
Materials
4 microcentrifuge tubes
2 Empty (clear/colorless)
CaCl2 on ice (blue)
either PM1 or PM2 on ice. (clear/colorless)
4 disposable transfer pipette
3 Inoculating loops
LB plates (2 LB/Amp - red line, 1 LB no Amp)
Lab station waste containers - one with 10% bleach, other empty
Equipment:
Vortex
Hot water bath
Incubator
42˚C for 45 seconds
37˚C / 98.6˚F
Human body temperature

5. Plate Streaking Techniques
Purpose is to spread out the bacteria so it has access to more food and space

6. Pipette Techniques Squeeze the bulb with two fingers - firmly - hold
Insert into the fluid
Gently release SOME pressure on the bulb! Until the liquid gets to the 0.1 m mark
Maintain the pressure
If you release all pressure the fluid will be sucked up into the bulb rather than to the measurement line.
Move the pipette to the microcentrifuge tube
Squeeze the fluid from the pipette into the tube.
Repeat, measuring to 0.25 mL
Add the liquid to the microcentrifuge tube
Repeat measuring 0.5 mL, then
Move 1.0 mL into the pipette then to the microcentrifuge tube
Cap, Mix with rocking & vortex, empty and returnL
1.0 mL
0.5 mL
0.25 mL
0.1 mL

7. Label your equipment Table + Table - LB/AMP + LB/AMP - LB/No AMP -
P5 Table ?
P5 Table ?
P5 Table ?
Table +
Table -

8. Transformation Flow Chart+ -
CaCl2 +
Bacteria
Plasmid
(PM1 or PM2)
CaCl2 +
Bacteria
LB/Amp +
LB/Amp -
LB/No Amp -
Purpose of
Each plate
Control Plate: LB/No Amp-:
“Lawn” testing viability
of the bacteria
and the nutrient
Experimental Plate:
Colonies of bacteria
With plasmids
Control Plate: LB/Amp-:
Testing effectiveness
Of Ampicillin
And contamination 8

9. Transformation Flow Chart+ -
CaCl2 +
Bacteria
Plasmid
(PM1 or PM2)
CaCl2 +
Bacteria
LB/Amp +
LB/Amp -
LB/No Amp - 9

10. Micropipette Techniques
Press and hold the black “set” button
Turn the dial
to the desired measurement in µ L
Attach the micropipette tip
Press the Yellow or Blue Plunger down to the stop
Insert the pipette tip into the substance
Release the plunger (the proper amount has now
been collected)
Position the tip over the microcentrifuge tube
to dispense, press the plunger to the stop
To eject the tip when done, press the plunger all the way down.

11. 1. Sign up with your partner at the lab table (determines)
To prep for the lab:
1. Sign up with your partner at the lab table (determines)
a. work station
b. partners sign up
c. which Plasmid Mix (PM 1 or PM2) you’ll have
d. create your hypothesis for your table
2. Practice techniques:
a. streaking
b. pipetting
3. Get & Label your materials
4. Complete the lab
PM means Plasmid Mix
PM1
Green
Blue
Grape
PM2
Cherry
Tangerine
Banana