Intro to Biotechnology Lab Notebook

A3-A4
Granite Technical Institute
Ally Mortensen
2015-2016

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Page # Title of Activity

3-4 Cheese Production

5-6 Molecular Structure
7-8 Measuring Small Volumes
9-11 Measuring Very Small Volumes
12-13 Measuring Mass
14-15 Molarity Solutions
16-17 Learning Spec
18-19 Mass/Volume Solutions
20-21 Measuring pH
22-24 Seriel Dilution
25-26 Agarose Gel
27-30 Lambda DNA
31-34 ELISA
35-37 Protein Fingerprint
38-39 Plates as Magic Glasses
40-42 Gram Stain
43-45 pGLO
46-47 GFP Purification
48-51 Plasmid Project

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Title: Cheese Production

Purpose: To determine whether or not the type of milk would affect the amount of
cheese curdled/whey produced during cheese production.

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Flow Chart:

Materials: 6 test tubes, 4 pipettes, pasteurized whole cow milk, pasteurized soy
milk, raw goats milk, 3 graduated cylinders, filter paper, 3 funnels, and vinegar

Procedure:
1. We gathered the materials needed to conduct the
experiment, and labeled the tubes.
2. Using pipettes, we then took the 6 test tubes and put each
type of milk (7 mL) into two tubes.
3. Next we pipetted 1.75 mL of vinegar into 3 of the test
tubes (one type of milk each).
4. We then began to invert all 6 test tubes three times,
slowly, and then placed all six tubes in a water bath for 15 minutes
and 37 C.
5. After the 15 minutes was up, we took the 3 graduated
cylinders, 3 funnels, and the filter paper and began to build our whey-
o-meter.
6. Finally, using the whey-o-meter, we measured the
amount of why and curds from the 3 test tubes containing vinegar.

Results: Our results were that the raw goats milk and the pasteurized whole
cows milk produced the same amount of whey (6 mL) while the pasteurized soy
milk only produced 5 mL of whey.

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Conclusion: After the experiment, we came to the conclusion that different types
of milk didnt quite affect the amount of why produced during cheese production.

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Title: Molecular Structure
Purpose: To determine whether or not bleach would have the same denaturing
effect on albumin as lemon juice.
Flow Chart:

Materials: 4 Pipettes, 4 test tubes, Two 50 mL graduated cylinders, Cheese Cloth,

Scale (g), Funnel, Water bath (99 C), 50 mL Egg Whites, 3 mL lemon juice, 3 mL
bleach, 3 mL distilled water
Procedure:
1. We began our experiment by gathering the necessary
materials, organizing them, and labeling them.
2. Next we added 50 mL of egg whtites into one of the 50
mL graduated cylinders, and the took that and added 3.5 mL of egg
whites into all four test tubes, and then added 1.5 mL of each pH into
its respected test tube, and leaving the fourth tube with only egg
whites.
3. After the liquids were placed accordingly into their
designated test tube, we inverted each tube three times, slowly, and

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 then placed all four test tubes into the water bath at 99 C for 5
minutes.
4. We then took the tubes out of the water bath and filtered
out the contents of the tubes using one of the 50 mL graduated
cylinders and some pieces of cheese cloth. By applying this process
the cheese cloth caught the denatured albumin and filtered out the
remaining liquid.
5. Next we took the filtered out denatured albumin and
measure the weight (in grams) on a scale.
6. Lastly we observed and recorded our results.

Results:

Conclusion: After conducting this experiment, we came to the conclusion that had
less of a denaturing fact on albumin than lemon juice, and distilled water had no

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affect at all. We also concluded that adding a pH produced less albumin than just
applying heat.

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Title: Measuring Small Volumes Lab
Purpose: To learn the functionality and purpose of pipettes and pipette pumps, in
milliliters.
Flow Chart:

Materials: 8 test tubes, red colored water, blue colored water, green colored water,
yellow colored water, purple colored water, 5 pipettes (10 mL), tube racks, small
plastic cups, Biotechnology Lab Manual
Procedure:
1. Organize and prepare the necessary materials for the experiment
2. Obtain the Biotechnology Lab manual and follow the written
procedure on pages 34-35
3. Begin the experiment, then observe and record the results
Results: After following the lab manuals procedure and conducting the
experiment, we took our finalized test tubes and compared them with the teachers
key test tubes. Our results were that our tubes were extremely close of not exactly
the same to the teachers key tubes.

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Figure 1: The first set of test tubes from the first Figure 2: The second set of test tubes from the second

experiment, next to the key tubes. Experiment next to the second key tubes.

Conclusion: In this experiment we learned how to use pipettes and pipette pumps
by following two pre-written procedures in the Biotechnology Lab Manual. After
doing this experiments, we concluded that our test tubes were the same as the key
tubes, and that we had obtained a thorough understanding of a pipette, how it
works, and what its used for.

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Title: Measuring Very Small Volumes
Purpose: To learn about and understand the functionality of a micropipette, how it
works, and what its used for. Also, the purpose of this lab is to learn how to
precisely measure small volume amounts.
Flow Chart:

Materials: 8 test tubes, micropipettes (0.5-10, 2-20, 20-200, 100-1000), test tube
rack, centrifuge, red colored water, blue colored water, green colored water,
Biotechnology Lab Manual, micropipette tips (appropriate sizes)
Procedure:
1. Obtain the necessary materials and organize accordingly.
2. Follow the pre-written procedure in the Biotechnology Lab Manual on
pages 37-38.
3. Start the experiment when all materials are prepared, and make sure to
follow the procedure.

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 4. Once the experiment is complete, observe and record data. Compare
your tubes with the teachers key tubes.
5. Finally, re-extract the liquid from the tube, with the correct
dial/measurement setting as instructed by the lab manual, and determine
whether or not your measurements were correct or slightly mistaken.
Results: After completing this experiment/procedure, we took our completed test
tubes ad compared them to the teachers key tubes. We found out that even though
we were almost certain we used the micropipettes correctly, we were still slightly
off on most of our measurements.

Procedure Measurement Instructions for First Set

Tube # Red Dye Blue Dye Green Dye Total Volume
(Microliters)
1 27.2 313.0 59.3 399.5
2 555.0 222.0 7.8 784.8

3 133.3 19.8 235.0 388.1

4 9.4 4.1 2.25 15.75

Procedure Measurement Instructions for Second Set

Tube Solution I Solution II Solutio Solution Solution V

Letter n III IV
A 4.0 5.0 2.0 --- ---

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 B 6.5 2.5 --- --- 2.0
C 22.3 31.6 --- 44.4 ---

D 253 --- --- --- 557

Final Volume Results

Test Tube Theo. Volume Observ. Volume Error %

A 11 11.2 1.81%
B 11 11.5 4.55%
C 98.3 98.6 .3%
D 810 740 -8.64%

1 399.5 405 1.36%

2 784.8 775 -1.25%

3 388.1 381 -1.83%

4 15.75 15.69 -0.83%

Conclusion: Based on the results from our experiment, we concluded that

although we have a basic understanding of how to use and handle a micropipette,
we still need some practice with the. According to our results, we either over
measured or under measured on all of our tubes. But at the end of the experiment,
we have an entirely better understanding of what a micropipette is and how its
used.

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Title: Measuring Mass Lab
Purpose: To measure glucose and learn how to make glucose solutions with a
specific volume.
Flow Chart:

Materials: Balance (analytical and tabletop milligram), weigh paper, weigh boat,
glucose, lab scoops, tubes (15 mL), tube racks, permanent markers, pipettes (10
mL), green pipette pump, glucose test strips
Procedure:
1. Gather necessary materials
2. Fill 4 test tubes with 5 mL of D water
3. Find the mg of the glucose and add to the water
4. Invert the tubes in order to dissolve the glucose in the D water
5. Fill the tubes the remaining 10mL with D water
6. Place the tubes into the vortex for a few minutes to thoroughly mix
the solution
7. Use glucose test strips to compare the color tabs to the concentration
Results:

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Conclusion: After conducting this experiment, we came to the conclusion that
using the pH strips didnt give a very accurate reading of the pH balance of the
liquids. However, this may have been due to the fact that the pictures of the strips
were not taking in a good time frame. Nonetheless, the strips did reveal to us that
the milligrams of glucose did seem to increase.

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Title: Molarity Solutions
Purpose: The purpose of this lab is to make five solutions of copper sulfate, each
with a different molarity concentration. It also allows us to practice the molarity
equation 1M=1 mole solute/1L solution (or Moe).
Flow Chart:

Materials:
Analytical Tube Racks Weigh Paper (4) Weigh Boat (1)
Balance
Lab Scoop Cupric Sulfate 5 Tubes (15 mL) Labeling Marker

Procedure:

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 1. We began the experiment by determining the amount of copper sulfate
that would be needed for each of the 5 solutions, using the molarity
equation.
2. Next we gathered the necessary materials and labeled them.
3. Then the actual experiment began as we filled up all 5 test tubes with
2 mL of distilled water, and started to sort out the different concentrations of
copper sulfate for its respective tube.
4. We procured the first tube, and added .8 mg of copper sulfate to the 2
mL of D-water, which created a 1.0 molarity concentration. Then we filled
the tube up to the 5 mL mark with D-water.
5. The second tube has a similar process as the first tube, except tube
two contained .4 mg of copper sulfate, which created a 0.5 molarity
concentration, and filled the rest up the tube up to 5 mL with distilled water.
6. Tube three contained .08 mg of copper sulfate, creating a 0.1 molarity
concentration, and was filled to 5 mL with D-water.
7. The next tube, tube four, contained 0.4 mg of copper sulfate to make a
0.05 molarity concentration, and filled the tube with D-water (to 5 mL).
8. The last tube measured out .008 mg of copper sulfate to create a 0.01
molarity concentration, and filled the tube up to 5 mL with D-water.
9. After inverting the tubes a few times, we then observed the different
colors and concentrations of the different copper sulfate solutions, and
recorded our results.
Results:
The results of this experiment show to be pretty clear and concise. The cupric
sulfate diluted extremely well in the solutions and created a very diverse variety of
color.

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Conclusion:
The ending results of this experiment yielded perfect dilutions using the molarity
equation. In the photos above, they demonstrate how the different concentrations
of the copper sulfate move from lighter to darker, displaying how the
concentrations can widely affect a solution dilution, and how easily it can be
observed. According to the equation, we successfully completed the experiment,
due to the fact that as the concentration got smaller, the color in the tubes got
lighter, and vice versa.

Title: Learning Spec

Purpose: The purpose of this lab is to observe different colors with different
wavelengths, and to understand and learn to correctly use a spectrophotometer and
what relationship it has with different light spectrums.
Flow Chart:

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Materials:
1 Spectrophotometer
Procedure:
1. After learning what the spectrophotometer is, how is functions, and its
purpose, have one person in your group be the observer and look into the
spectrophotometer, announcing the colors as they see them.
2. Have another person in your group turn the wavelength dial, which
displays the numbers that represent the wavelength at which certain colors
appear.
3. When the first person announces the color they see, the second person
will stop turning the knob, and observe the number displayed. They then
announce the number displayed to the third person in the group.
4. The third person will be recording the data as it is announced on a
chart.
5. Have the entire class do this, and then after all groups complete the
lab, compare all of the data and find the percent error using the equation
Error=(actual)(expected)/(expected)x100

Results:
After the entire class had used the spectrophotometer and everyones results were
written on the white board, the results were compared and analyzed. Although all
of the numbers were different, they were all close enough together to be considered
valid answers. We also found that the wavelengths that we received were very
close to the expected wavelengths, which proves our answers to be fairly accurate.

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Conclusion:
After this experiment, we were able to correctly use the spectrophotometer and
describe what its purpose is. After looking at the entire class results, we came to
the conclusion that every single person sees light different and at different
spectrums, which also means that no one person sees a color the same way as
others. This conclusion is proof that every single human being thinks similarly, but
not exactly the same, which shows that everyone really is different and unique.

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Title: Mass/Volume Solutions
Purpose: The purpose of this lab is to able to make solutions, put them into the
spectrophotometer, and be able to observe the different wavelengths of light, and to
see the red, orange, yellow, green, blue, indigo, and violet colors in the
spectrophotometer.
Flow Chart:

Materials:
Spectrophotometer Distilled Cuvettes 5 test tubes Weigh
Water paper(5)
Analytical Balance Lab Scoop Copper Vortex Kimwipes
sulfate

Procedure:
1. First, we gathered the necessary materials for this experiment, labeled,
and organized them.
2. Next, we filled up all 5 tubes halfway with distilled water, and
obtained our analytical balance.
3. After getting the balance, we set a piece of weigh paper on the
balance, zeroed it out, and use the lab scoop to scoop little piles of copper
sulfate onto the balance until we reached our desired amount for each tube.

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 4. Once each desired amount was obtained, the different mg of copper
sulfate was placed into its respective tubes, and filled the rest of the tubes up
with its specific amount of needed D-water.
5. The tubes were then placed onto a vortex in order to make sure the
copper sulfate had completely mixed in with the D-water, and everything
was completely dissolved.
6. Next, we placed our samples into different cuvettes, and ran them
through the spectrophotometer, setting the wavelength to 590, the
absorbance to 0.0, and then the percent transmittance to 0.0 for each tube.
7. The samples were then run through and observed, and the results
recorded.
Results:
The results of this lab are shown in the table below, which demonstrates as the
concentration of the sample increases, the absorbance level goes up, while the %
transmittance decreases.
Sample Concentration Absorbance % Transmittance
300mg/mL 1.140 7.2
150mg/mL 0.528 29.6
75mg/mL 0.276 53.0
37.5mg/mL 0.119 76.0
18.75mg/mL 0.7 85.2

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Conclusion:
After completing this lab, we came to the conclusion that due to the different
colors given off by our different copper sulfate solutions, different wavelengths of
light were produced.
Title: Measuring pH
Purpose: The purpose of this lab is to learn how to identify and use a pH scale, pH
meter, and pH strips. We will also test 16 different variables and determine whether
their pH is acidic or basic, determined by the pH scale.
Flow Chart:

Materials:

Grape Juice Cleaner Apple Juice Milk

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Mountain Dew Cappuccino Monster Energy Vinegar
HPl Mango DH2O Bleach
NaOH Coffee Spic-n-Span Spring Water
pH Meter pH Strips

Procedure:
1. Obtain all of the necessary materials listed above, and make sure they
have some sort of label so that they are identifiable.
2. Set up the pH meter and lay out all of the pH strips in order from
acidic to neutral to basic.
3. Each person in the class will choose two variables to measure; one
will be done using the pH meter while the other is done with the strips. Be
sure to not choose the same variable for each testing material.
4. After the class has tested their variables and obtained their results, a
chart is made on the white board, and each student will record their results
within the chart.
5. As a class, come to a conclusion as to whether or not the variables that
were tested were more acidic, more basic, or neutral on the pH scale.
Results:
The results of this lab showed that different variables contained different pH levels
based on their ingredients and concentrations. The pictures below show both the
class results, and my personal results with testing the Mtn. Dew.

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Conclusion:

Conclusion:
After completing this lab, we came to the conclusion that different liquids differ in
pH all over the scale. We learned the difference between the strips and the meter
and now know how to correctly use both. After going over the class chart, we all
came to a similar agreement on all of our results and where the variables would
settle on the pH scale. However, some students failed to right an accurate number
on the chart seen above, so some of the results are not completely spot on, but they
were still close enough to other students results that they were still useable.

Title: Serial Dilutions

Purpose: The purpose of this lab is to be able to make several solutions and then
to successfully dilute them, and then to observe both the absorbance and percent
transmittance of the solutions using the spectrophotometer.
Flow Chart:

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Materials:
Strawberry 5 Spec Cuvettes Spectrophotometer 5 Test tubes
Daiquiri Sobe
Vortex Pipettes Kim Wipes DH2O

Procedure:
1. Gather, label, and organize necessary materials.
2. Come up with a concentration to dilute the variable.
3. Once the dilution ratio is acquired, each test tube will be filled with
the same amount of Sobe (5mL).
4. Next, the dilution begins. Each tube will be diluted more and more,
based on the equation that we used to come up with the dilution
concentration.
5. After each tube has become diluted with the distilled water, place the
tubes into a vortex and mix well to ensure that the solution is completely
diluted.
6. Next, take 5 mL of each tube and place them into clean
spectrophotometer cuvettes. Obtain a cuvette filled with distilled water, and
place that first in the spectrophotometer.
7. Set the absorbance and the percent transmittance to 0.0, and remove
the distilled water cuvette (the blank). Before putting and other cuvette into
the spectrophotometer, wipe the tube down with Kim wipes each time.

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 8. Place the cuvettes into the spectrophotometer one at a time, and
observe and record the solutions absorbance and percent transmittance.

Results:
After the solutions of the Sobe were completely diluted and place in the
spectrophotometer, the results yielded that as the concentration of the Sobe got
smaller, the absorbance decreased while the transmittance increased.
Dilution Concentration Absorbance % Transmittance
1x 1.999+ 0.8
.5x 1.580 2.6
.25x .905 12.4
.125x .494 32.0

Conclusion:
A dilution is when a substance (usually a liquid) is diluted with another substance
(usually water), which creates a concentration decrease within the original liquid

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substance that was used. With a differed dilution, the substance becomes less and
less concentrated as more of the dilution variable is added to the original
substance.
When putting the diluted solutions into a spectrophotometer, it appears as the
concentration becomes lower, the absorbance also lowers. However, as the
concentration decreases within the substance, the percent transmittance increases.
This essentially means that as the solution created becomes more diluted, the
amount of light that the solution can absorb also decreases, while the amount of
light transmittance going through the solution increases. As the dilution continues,
the solutions become clearer and clearer to see through.

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Title: Agarose Gel
Purpose: The purpose of this lab is to learn how correctly produce an agarose gel,
that will later be used for DNA electrophoresis.
Flow Chart:

Materials:
Tape Agarose powder 50 mL grad. Cylinder
Small flask Plastic bag Electrophoresis box
Microwave Buffer solution Paper towel
Heating pads

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Procedure:
1. Observe the demonstration of how to make an agarose given by the
instructor.
2. Gather necessary materials; label and organize them.
3. Prepare a buffer solution of 2,940 mL of water and 60 mL of buffer,
giving a total of 3,000 mL of buffer solution.
4. Prepare the agarose powder by obtaining 1 gram of powder, and mix it
with 50 mL of buffer solution.
5. Mix the agarose-buffer solution in a small flask, and place a paper
towel in the opening of the flask to prevent spilling.
6. Place the flask in the microwave, and start the microwave for about 3
minutes. Carefully watch the flask while it remains in the microwave, and
remove the flask when the solution begins to boil.
7. Repeat step 6 as many times as necessary until the solution shows to
be entirely clear. Be sure to use heating pads or gloves when removing the
flask, as the solution will be hot.
8. Allow the solution to cool to about room temperature.
9. While the solution is cooling, prepare the agarose box. Tightly tape
both end of the box, so that when the agarose is poured into the box nothing
will leak out.
10. Once the solution has cooled, pour it into the prepared agarose box,
with the comb sitting in the box. Allow the gel to cool and harden.
11. Finally, after the gel has cooled, place it in a zip-lock back and leave
in the fridge over night.
12. Take out of fridge the next day, and observe results.
Results: The result of this experiment was a well-prepared agarose gel. The gel
was made and cooled properly, which allowed it to be ready to use with
electrophoresis DNA running.

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Conclusion:
After conducting this experiment, we found that the agarose was well prepared and
ready for the electrophoresis process. We came to understand how exactly to
prepare an agarose gel, what its used for, and how it is supposed to look when
done correctly. The agarose gel looked as though it was ready for electrophoresis
and to run DNA, and we had successfully created an electrophoresis agarose gel.

Title: Lambda DNA

Purpose: The purpose of this lab is to learn how to correctly use an agarose gel to
run DNA, and to learn how to cut up DNA using several different restriction
enzymes.
Flow Chart:

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Materials:
Electrophoresis Lambda Virus Marker Restriction Buffer
Box DNA
EcoRI HindIII PstI Agarose Gel

Water bath Ice Centrifuge Fridge

DNA Stain 0.5-10 l Micropipette tips Micro test tubes

Micropipette (4)
1x TAE Buffer

Procedure:
1. Begin by obtaining the Lambda DNA, all 3 Restriction enzymes, and
the restriction buffer from the instructor. Keep the DNA and the restriction
buffer on ice.

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 2. Label the micro test tubes in order with an initial representing what
the tubes will contain (ex: L, P, E, H)
3. Next, grab the micropipette and a fresh tip, and begin pipetting 4
micro liters of DNA into EACH tube. Then, with a fresh tip, apply 6 micro
liters of R buffer into the tube labeled L ONLY. With the last 3 tubes, apply 5
micro liters of buffer.
4. Then, obtain the tube labeled P, and with a fresh micropipette tip,
apply 1 micro liter of PstI. Pipette mix the solution, close the lid, and set it in
the ice.
5. Grab the tube labeled E, and using a fresh micropipette tip, apply 1
micro liter of EcoRI into the tube, pipette mix, close the lid and place the
tube in the ice.
6. Obtain the final tube labeled H, and a fresh micropipette tip. Place 1
micro liters of HindIII into the tube, pipette mix, close the lid, and place in
ice. Next grab all four tubes, and place them into the centrifuge, making sure
to place the tubes apart evenly. Spin the tubes for a few seconds, until all of
the solution is down at the bottom of the tube.
7. Take the tubes out of the centrifuge, and place them in a water bath set
at 37 degrees Celsius for 30 minutes, and then place in the fridge overnight.
8. The next day, take the samples from the fridge and add 2 micro liters
of loading dye to each tube.
9. Obtain the agarose gel from the fridge and place it into the
electrophoresis box with the wells closest to the negative side.
10.Next, take 10 micro liters of each sample and pipette them into their
own well in the agarose gel. Be sure to be careful and accurate, and try not
to bump the gel too much with the pipette tip.
11.Place the lid on the electrophoresis box, and turn the power on. Run
the machine at 100 volts for 40 minutes.
12.Once the electrophoresis is complete, carefully remove the gel from
the box and place it into a clean container. Add DNA stain to the container,
just enough so that a thin layer covers the gel.

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 13. Stain the gel for 12-15 minutes, gently rocking the entire time. Once
this time is up. Place the stain back into the jar and begin the de-staining
process.
14.Begin the overnight de-staining process by placing the gel under
running water in tiers upon other gels. Leave under lukewarm running water
overnight.
15.The next day, air dry the gel and observe the DNA bands in the gel,
and record the results.

Results: The results of the DNA within the agarose gel for this run were
inconclusive due to the DNA stain being expired without our knowledge at the
time, inconclusive meaning that only one band in one well was visible, although
barely.
The same experiment was ran a second time, with the exact same procedure,
except this run used a 2x concentration instead of a 3x in an attempt to make the
DNA in the agarose move farther and be slightly more visible. The second run
yielded much better results than the first, displaying all 5 strains of DNA. A newer
DNA stain was used in the staining process, which stuck to the DNA considerably
better than the first run, and the DNA was thus visible in the agarose gel.
This experiment resulted in different restriction enzymes cutting the same Lambda
DNA virus in different places, and after running this experiment we found that our
original DNA strain (M) was cut with HindIII, as it showed the same DNA cut
sequence as the well containing HindIII and Lambda.

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Conclusion: The purpose of an agarose gel is to run DNA through the gel using
the process of electrophoresis, which when done correctly will display DNA strains
that have moved through the gel from electricity pushing it.

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Lambda DNA is a bacteriophage, and is a virus that is harmless to humans which
makes it a great demonstrative virus DNA to use in experimenting with DNA
sequences and restriction enzymes. A restriction enzyme is used to cut up DNA in
different areas of the base pairs, which is what allows every person and living
being to be as unique as it is. When different restriction enzymes are used in the
same DNA strain, they will cut up the DNA in different areas, resulting in different
new strains of that specific DNA. This allows us to see the different effects that
restriction enzymes have on viruses and other things.
After conducting this lab, we have come to the conclusion that different restriction
enzymes paired with the same strains of DNA will result in different cut sites
within the DNA.

Title: ELISA Lab

Purpose: To understand the purpose of ELISA, and to identify how to work
backwards from the results to reveal Patient Zero.
Flow Chart:

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 Materials:
1 bodily fluid 20-200 L 1 pipette Wells (9)
tube Micropipette
Wash Buffer (70- Positive Control Negative Control Primary Antibody
80 mL) (PA)
Secondary Enzyme Substrate Pipette Tips Large stack of
Antibody (SA) (SUB) paper towels

Procedure:
1. Obtain a bodily fluid sample with a micropipette, and identify the
number on the tube.
2. Create a chart with up to 23 columns, and 3 rows.
3. Begin trial 1 by swapping half of your bodily fluid with another
person.
4. Write your number under the number you switched with and vice
versa on the chart.
5. For the second trial, swap half of your bodily fluid with another,
different person. Write their number under yours and vice versa on the chart.
6. For the third and final trial, swap half of your bodily fluid one last
time with another, different person. Write their number under yours and vice
versa on the chart.

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 7. Obtain the different proteins (PA, SA, and SUB) from the instructor.
Label your wells with 3 positive controls, 3 negative controls, and 3 of your
sample, labeled with the number on the tube you received.
8. Using a fresh pipette tip, and the 20-200 microliter pipette, transfer 50
microliters on the positive control into each of the three positive control
wells.
9. Using a fresh pipette tip, transfer 50 microliters of the negative control
into the wells labeled with negative control.
10. Using a fresh pipette tip, transfer 50 microliters of your sample into
the appropriately labeled wells. Wait 5 minutes for all of the proteins to bind
to the plastic wells, then begin the wash process.
11. Begin the wash process by dumping out all of the liquids in the wells
onto a stack of paper towels, begin sure that all of the liquid is out. Then, fill
each well with wash buffer with the designated plastic pipette. Dump the
wells onto a fresh stack of paper towels, and then discard the towels.
12. Repeat this was process twice.
13.Use a fresh pipette tip to transfer 50 microliters of PA into all 9 wells,
and then wait 5 minutes for the proteins to bind to the plastic. Begin the
wash process, and repeat it twice.
14. Obtain a fresh micropipette tip once more, and fill all 9 wells with
SA, wait five minutes, and then repeat the wash process for a final time,
repeating it 3 times.
15. Use a fresh pipette tip and transfer 50 microliters of SUB into all 9
wells. Wait 5 five minutes, then observe and record results.

Results:
After the entire class had conducted this experiment and revealed their results, we
came to find that a good number of students had become infected after the sharing
process. However, due to cross contamination, some students results were
inconclusive, and therefore unusable. Nonetheless, we (as a class) were able to

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gather enough information to begin to determine who patient zero could have been.
Not knowing who it was to begin with, and whom that person shared with in round
one, there is no possible way to narrow it down to one single person, though we
can narrow it down to two. After working backwards with our available results, we
were able to conclude that patient zero was either

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Conclusion:
All diseases or viruses begin with patient zero. As patient zero comes into contact
with others, that person is transferring the disease onto those people, causing the
infection to spread. AS the infection spreads and more and more people become
infected and the virus becomes more known, scientists begin to gather information
about the new disease using the ELISA method, which allows the scientist to
determine who has become infected and, with enough information, can work their
way backward using their results to determine who patient zero might have been.
This process allows more information to be revealed about the new disease and
allows us to try to begin to find a cure for it. The more we know about the person
who contracted the new illness, the easier it becomes for us to find a cure.

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Title: Protein Fingerprint
Purpose: To learn and understand the process of running proteins through a
polyacrylamide gel using the concept of electrophoresis.
Flow Chart:

Materials:
Halibut Tuna Salmon Bass
Mahi mahi 5 flip top micro 5 screw top micro 20-200l
tubes tubes Micropipette
Water bath 0.5-10l PPP Kaleidoscope Actin and Myosin

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 Micropipette Standard standard
1x TGE buffer Ready gel cassette Mini-PROTEAN
gel box

Procedure:
1. Gather the necessary materials and begin labeling each flip top micro
test tube with each different fish sample. Write the sample initials on the lids
2. Add 250 micro liters of Bio-Rad Laemmli sample buffer to each of
the flip top tubes.
3. Cut a small piece of fish from each sample and place the sample into
the respective tube. Close the lids, and then fish the tubes about 15 times in
order to agitate the fish sample tissue with the buffer.
4. Incubate the tubes for 5 minutes at room temperature, and then
carefully transfer the buffer from each sample into the screw top tubes,
making sure to label them correctly on the side of the tubes. Do NOT
transfer the fish samples.
5. Place the buffer samples in the screw cap tubes in the water bath at
95 C for 5 minutes.
6. During this five minutes, prepare the gel box with the Ready Gel
cassette by placing the cassette into the electrode assembly with the short
plate facing inward. Place another Ready Gel cassette on the opposite side of
the electrode, with the notch buffer dam facing inward.
7. Slide the buffer dam, the cassette, and the electrode assembly into the
clamping frame.
8. Press down the electrode assembly while closing the two cam levers
of the clamping frame.
9. Lower the inner chamber into the mini tank, and then completely fill
the box with the 1x TGE buffer.

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 10. Begin loading the gel by leaving well 1 and 2 blank, and loading well
3 with 5 micro liters of the PPP Kaleidoscope standard.
11. Next, fill wells 4-8 with 10 micro liters of each sample in its own
well, making sure to use a fresh micro pipette tip with each different sample
loading.
12. Run the gel using electrophoresis for 30 minutes at 200v, and then
remove the gel from the cassette and begin the gel staining process, staining
for about 1 hour while gently shaking for best results.
13. Discard the stain dye, and destain the gel in a large volume of water
overnight. Dry the gels using GelAir cellophane. Observe and record results.

Results:
As depicted in the photo below the results came out to be kind of mashed together;
the bands dont create very distinct lines, making it difficult to tell the different
molecule bands apart. Due to this unfortunate turnout, we had to mark these results
as inconclusive.

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Conclusion:
After completing this lab and reading through the background information, we
have come to the conclusion that species can be related through the process of
evolution. Using the PAGE process is an excellent way to run different proteins
from different organisms through an agarose gel and, using electrophoresis,
determining which organisms are more closely related depending on the
similarities between the bands of protein.

Title: Plates as Magic Glasses

Purpose: To understand and demonstrate how to correctly swab and plate bacteria
that will later be observed.
Flow Chart:

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 Materials:
1 Petri Dish 4 sterile Sharpie Agar Incubator
swabs

Procedure:
1. Begin by gathering all necessary materials and dividing the Petri Dish
into quadrants, making sure to NOT write on the lid, but on the bottom of
the plate.
2. Label one quadrant as the positive control, the second quadrant as the
negative control, the third quadrant as the mouth bacteria, and the fourth
quadrant as the bacteria of your choice.
3. Then we received the positive control swab, and swabbed it on the
positive control quadrant of the plate. We left the negative control quadrant
alone, as we did not want any bacteria to grow on that quadrant.
4. Next we took a sterile swab out of its package very carefully, as to
keep it as sterile as possible, and swabbed the top of our tongues. After
swabbing, we wiped the swab all over the quadrant labeled mouth.
5. For the last quadrant we were allowed to swab any surface within the
school that we like, and I swabbed the stairs door handle on the first floor,
and swabbed that onto the correct quadrant.
6. Lastly, we placed the lid on the petri dish, flipped it upside down, and
placed our bacteria in the incubator to grow.
Results:

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 After the incubation process and allowing the bacteria to grow, the results of
the growing process were tiny little blots of yellow. These little blots of yellow are
the bacteria that were grown, and most were grown in little colonies, or groups.

Conclusion:
After completing this lab, Ive come to the conclusion that bacteria can be very
easily grown and very easily acquired when done correctly. Since bacteria is very
easy to grow, use, and obtain, its a prime candidate for a vast amount of different
experiments. From this lab Ive learned that bacteria grows on plates after being
incubated for at least a day or so, and will grow either isolated, or in little
groupings known as colonies. However, in order to grow on the Petri dishes, it also
needs a layer of agar, as it requires something to grow on and stick too. Just as
well, the plates are tipped upside down during incubation, as bacteria like to grow
on the ceiling of things.

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 Title: Gram Stain
Purpose: To demonstrate and understand how to essentially dye bacteria so as to
be able to physically see the singular cells under the microscope, and determine by
color whether to not the sample is gram positive or gram negative.
Flow Chart:

Materials:
Cultured Inoculation Distilled Clean slide Flame
Bacteria loop water
Ammonium Iodine Kim wipes 95% Ethanol Safranin
oxalate violet counterstain
stain
Microscope Regular water

Procedure:
1. We began this experiment by gathering all necessary materials,
including our previously cultured bacteria, and adding a drop of distilled
water to a clean and dry glass slide.
2. Next, we acquired a sterile inoculation loops, and transferred a tiny bit
of our mouth bacteria into the drop of distilled water, which we then mixed
into a thin film.

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 3. Afterwards, we waved the slide over a flame in order to quickly dry
up the water/bacteria mix, making sure to not hold it over the flame the
entire time, as that would cause the glass to break.
4. Once the drop had completely dried up we began the process of
Primary Staining, in which we covered the little dip in the slide with a drop
or two of Huckers ammonium oxalate crystal violet stain, and let it sit for
approximately one minute.
5. After 1 minute, we carefully rinsed the slide with gently flowing
water, and then added a drop of gram iodine solution, and let that sit for one
minute as well.
6. The next step was to decolorize for 30 seconds by adding 95% ethanol
and agitation to the slide, mainly by flicking it with our finger. Then we
gently blotted the slide dry with a clean kim wipe.
7. Next we moved onto counter staining, and we started by adding
safranin counterstain to the slide for approximately 1 minute, rinse with
gently flowing water, and then blot dry with a kim wipe and let stand to air
dry.
8. The last step was to observe our freshly stained bacteria by placing the
slide underneath the microscope lens to observe the color and shape of the
cells, and record our observations and results.

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Results:

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After completing this lab and observing my plate through the microscope lens, I
found my bacteria to be a pink color in small circular chains, which resulted in my
cells being gram negative, which means this bacteria is relatively harmless to most
humans.

Conclusion:
After completing this lab, Ive come to the conclusion that bacteria is more easily
seen when stained, as it brings out the color and the shape a lot more clearly.
However, since there are several different types of bacteria, they all have different
colors and generally different shapes. All of these different colors and shapes
represent different types and strains of bacteria, which also generally help organize
and label which bacterias are harmful to human beings, which ones are not, and
which ones are actually helpful in keeping our bodies healthy. In my results, I
came to the conclusion that the bacteria from my mouth was plentiful, but since it
was pink in color and in small circular chains, it came to be gram negative, which
under usual circumstances means that it is not harmful to humans.

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 Title: pGLO
Purpose: To understand and be able to take different genes from other organisms
and insert them into a DNA plasmid in order to complete the process of genetic
engineering.
Flow Chart:

Materials:
4 culture plates LB Broth Inoculation loops Micropipette (100-
1000 L)

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Ice 2 Micro tubes E.coli bacteria CaCl2
transformation
solution
pGLO Plasmid

Procedure:
1. We began the experiment by collecting the necessary materials,
organizing them properly, and labeling them as needed.
2. After labeling the plates with the necessary information, we filled
each of the four culture plates about full with LB broth, and allowed the
broth to settle over a span of about 2 days.
3. Next we labeled 2 micro test tubes, one with +pGLO and one with
pGLO, and then added 250L of transformation fluid into the tubes, and
finally placed the tubes on ice.
4. Then we took a sterilized loop, obtained bacteria from a starter plate,
and added the bacteria to the positive pGLO tube, spinning the loop between
our fingers to ensure that the bacteria had been transferred into the tube.
5. The tube was then incubated on ice for 10 minutes, and then did an
immediate heat shock, in which we took the tubes off of the ice, immediately
placed them into a water bath at 42 C at exactly 50 seconds, and
immediately placed the tubes back onto the ice for 2 minutes.
6. Next we added 250L of LB Broth into each tube, using a fresh
pipette tip each time.
7. After the broth had settled and the plates were ready for bacteria, we
organized our plates according to what went on each plate, and began the
process of adding bacteria to the plates using the streaking method.
8. We obtained the bacteria from a starter plate using a sterilized
inoculation loop, and placed the bacteria on the plate in a streaking pattern.
9. We continued this process until all the plates that needed bacteria had
it, continuously sterilizing the loops in an open flame after streaking each
plate.

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 10. After streaking the bacteria onto the plates, we taped them together,
and placed them upside down to incubate in the incubator for about 2 days.
11.The next class period we removed the plates from the incubator, and
took them into a dark room and observed them under UV light to see the
glowing plasmids at work.

Results:
My results came to be inconclusive, as I made a mistake somewhere during the
process of the experiment, most likely during the heat shock period. In the end, my
bacteria did not end up glowing, as the pGLO plasmid did not successfully insert
itself into the bacteria.

Conclusion:
During this experiment we came to understand the process of how genetic
engineering is used to create general mutations that can be helpful or necessary for
the life of a human or animal. We learned how to isolate a plasmid and insert it into
a strand of DNA in order to genetically engineer something, for example human
insulin. By seeing our bacteria glow, we knew that the pGLO Plasmid had been

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successfully inserted into the DNA of the bacteria that we cultured. However, for
those whose bacteria did not glow, we know that there was a mistake made
somewhere during the process, and that the plasmid was no successfully inserted
through the visible evidence.

Title: GFP Purification

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Purpose: To purify the Green Fluorescent Protein (GFP) that we obtained from the
genetically engineered pGLO bacteria, using a column.
Flow Chart:

Materials:
Chromatography Column pGLO Bacteria TE Buffer

Lysis Buffer (10 L) Wash Buffer UV Light

Sterile Loop Ice Micro tubes (2)
Incubator

Procedure:
1. We began the experiment by creating two cultured plates. We did this
by spreading two mixtures of transformation broth on each plate, one
containing DNA and the other with no DNA
2. Next, as a class, we incubated the plates for 2 days, and then checked
the plates for glowing colonies
3. As a result, my plates did not yield any colonies, as I must have made
a mistake during the procedure of the last lab. (pGLO lab)

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 4. In order to continue this experiment, I borrowed some bacteria from
Brielle Koss, as her previous experiment was a success and her bacteria
glowed with the pGLO gene
5. Taking this borrowed bacteria; I used a sterile loop to extract the
bacteria and stirred it into a growth media, spinning the loop as to ensure the
bacteria mixes into the media
6. We then incubated this growth media, and afterwards very carefully
transferred the Green Florescent Protein (GFP) into a new tube
7. Next, we obtained a column, and dumped everything out of it besides
the pellet, and then added TE Buffer and 10 mL of lysis buffer before
freezing them
8. Afterwards, we cleansed the chromatography column to allow for the
final process of purification to occur
9. Lastly, after the final purification process had occurred, we rinsed the
column with wash buffer, and observed and recorded our results.
Results:
As I had borrowed some glowing bacteria from the previous project from Brielle
Koss, the purification process of this experiment was a success, as I had correctly
purified the gene due to the liquid glowing bright green when under UV light

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Conclusion:
After conducting this experiment I found that I now understand how to
successfully purify a gene from a genetically engineered strand of DNA with a
specific plasmid inserted into it; in this case the Green Florescent Protein from the
pGLO gene, obtained from the previous experiment. Just as well, I now also
understand how to correctly use a chromatography column in order to conduct the
process of purifying a gene from DNA. Furthermore, the ending liquid did glow
green under UV light, showing that the experiment was a success and the gene was
correctly purified.
Title: Plasmid Project
Purpose: To understand how to find and properly identify an unknown plasmid,
and prove that we understand how to make 20x TAE buffer and dilute it, make an
agarose gel, run a restriction digest, and run the gel using electrophoresis
Flow Chart:

Materials:
0.5-10 L Ice Water bath 3 micro tubes Centrifuge
Micropipette (37 C)
Lambda HindIII PstI EcoRI 0.5-10L
HindIII Micropipette

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 tips
Online NEB Online Virtual Tris base EDTA D Water
Cutter Digest
pH Strips Electrophoresi Agarose 1000mL Small beaker
s box bottle w/ lid
Paper towels Glacial acetic Fume hood Mystery
acid plasmid

Procedure:
1. I began this project by receiving my mystery plasmid from my
instructor, with the code.
2. Next I determined which three enzymes I was going to use for
this experiment. After performing a virtual digest using the three possible
plasmids (pKAN, pAMP, and pBLU) and the online NEB cutter, I found
that the experiment would work best using the enzymes EcoRI, PstI, and
HindII
3. I came to the conclusion that I would use EcoRI and PstI for my
double digest, and HindIII for my single
4. After obtaining this information, I began to make my 20x TAE
buffer. I used the equation Moe, and plugged in all of my numbers, and
found that I would need 19.38g of Tris Base in order to make a .8M
solution.
5. Next I used the Curly equation to find that in order to create a
0.5M EDTA solution, I would need 10 mL of EDTA.
6. After that, I added 4.6 mL of glacial acetic acid to my solution,
remaining under the fume hood while doing so. Finally I mixed the
solution, added d-water to adjust the final volume to 200 mL, and
determined that the pH of the solution was about 9.5 of the pH scale, using
pH strips.

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 7. After I had completed my buffer and properly labeled and
stored it, I began to make my agarose gel. I did this by Diluting my 20x
TAE to a 1X concentration, using the Moe equation, and knowing that I
wanted my gel to be a 1.4%, I came to the conclusion that I would need
about .7g of agarose for my gel. I mixed the solution, heated it until it was
clear, and poured it into the taped box and waited for the gel to solidify.
8. While waiting for my gel to solidify, I began to run my digest. I
began with my marker, placing 8L of Lambda cut with HindIII into a
micro tube. Next I did my single digest, in which I placed 1L of HindIII
into a tube, along with 5 of restriction buffer, and 4L of my mystery
plasmid. Lastly I did my double digest, in which I placed 1L of PstI and
1L of EcoRI into a tube, 5L of restriction buffer with about 4L of my
mystery plasmid.
9. After wards, I added 2L of a blue loading dye to each tube,
quickly centrifuged them, and placed all three tubes in the water bath for
about 30 minutes at 37 C.
10. After the 30 minutes, I took my tubes out of the water bath and
began the process of electrophoresis. I placed my completed gel into the
electrophoresis box, filled it to cover the gel with a 1x TAE buffer, and
began to fill the wells. The first lane was my marker, in which I placed
10L of it into the well. Lane 2 was my single digest, in which I placed
10L of my single digest into the well. My 3rd lane was my double digest,
in which I placed 10L of the digest into the well.
11. I placed the lid ob the box, making sure the wells were closest
to the negative poles, and ran the gen for 45 minutes at 100 volts.
12. After the process of electrophoresis was completed, I carefully
removed my gel from the box, placed it in a labeled Tupperware bowl, and
dyed it with purple gel stain. After it had stained, I let the gel sit in a plastic
bag filled with just a little bit of buffer in the refrigerator for about 48
hours.

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 13. After those 48 hours, I removed the gel from the bag, and began
the detaining process, in which I let the gel sit under a very light stream of
water for about an hour and a half, as to remove the stain and allow me to
see the results of my bands.
14. Once my gel had become de-stained, I removed it from the
sink and laced it on a light table to see the position of my bands. I then
observed and recorded my results.
ONLINE DIGEST RESULTS
pAMP pKAN pBLU
EcoRI 2137 EcoRI 2116 EcoRI 3139
PstI 946 PstI N/A PstI N/A
HindIII 233 HindIII 234 HindIII 3190
Plasmid Size: 4539 Plasmid Size: 4194 Plasmid Size: 5437

Results: After receiving my results from the physical gel and comparing them to
the results of my online digest, I have come to the conclusion that my mystery
plasmid was pBLU.

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Conclusion:
After conducting this experiment and working through the many processes I have
learned through out the year, I have been able to successfully make buffer, make a
gel, run a digest, and identify a plasmid that was unknown when given to me. This
experiment has allowed me to prove my skills that I have learned and acquired this
year and show exactly how to use them. This experiment has taught me how a day
to day life in the lab is when this is applied to a career, and taught me exactly what
will be done if I chose to work as a biotechnician of some sort in my future. Not
only have I obtained the results in a well and timely manner, but I have created
more confidence in being able to work through these processes quickly and
completely.

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