Holli Brown

CILI-CURE 21

02/28/2019

Objectives:

The objectives for this lab were for students to comprehend what PCR is, the different steps behind it, and the components that make it up. Students will also understand how primers work, and why we chose the 18SV4 primer. Students will work as a group to prepare samples for PCR analysis, as well as practice aseptic techniques to avoid contamination from their human DNA. Calculations will also be performed in order to approximate how much DNA should be added to the test solution, which will practice dilution skills gained last semester. Students will also begin thinking about presentations for the upcoming labs, and theorize how they can organize a poster to showcase their findings and data sets. Finally, students will review for the midterm, and recall all the knowledge they’ve gained from the last 8 months in CILI-CURE.

Methods and Lab Summary:

1. To begin lab, we will learn about the PCR procedure and our primers chosen in lab. Take notes in order to prepare for the Midterm next week, and to explain questions to others during the symposium poster presentations. As a scientist, it is important to know what you are talking about, and to be able to explain findings in simpler terms to others around you.
2. As a reminder, through the duration of this lab, ALWAYS wear gloves when handling samples. We want to avoid human DNA integration into the media, as the 18SV4 primers will also pick up human DNA and ruin the data when analyzed with PCR.
3. Before collecting material, wipe the lab benches down with a 10% bleach solution, and clean up with paper towels. This is to practice aseptic techniques and to eliminate any possible continents on the lab bench surfaces.
4. To begin, obtain an ice basin, and Eppendorf tubes for DI water, pDNA, primer, and an empty tube for dilution. Place these all on ice in order to maintain the integrity of the samples.
5. For the experimental portion of lab, we will start by preparing samples for PCR analysis. First, calculate how much of the pDNA is needed, and if dilutions need to be made. We want our drop to be less than 100 ng in order for the PCR analysis to work.
6. Create a dilution. Do this by adding no less than a 1 uL drop into an about of water that will create a dilution. For example, if your sample from the nano drop test was 600 ng/uL, dilute the sample by adding 1 uL of pDNA to 9 uL of pure DI water, to create a 1:10 dilution.
7. From the dilution, pipette a set amount into the empty Eppendorf tube, and label it ‘dilution’. In the example above, 1 uL of diluted DNA sample was placed into the test tube for a 60 ng/uL sample, which is below 100 ng/uL.
8. Next, we will create a test and control sample. Obtain 2 small tubes filled with the 12.5 uL of master mix. The master mix is made of TAC polymerase and DNA nucleotides.
9. For the test tube, place the specified amount of pDNA sample into one of the tubes. From this, calculations can be made of how much water and primer to add. For example, for a 1 uL drop of pDNA and 12.5 uL, 10.5 uL of water and 1 uL of primer should be added. If confused, see the chart below.
10. As a general rule, we always want to pipette with a p10 micropipette. With the p10, always have more than 1 uL, but less then 10 uL, as this will lessen the impact of human error. We use a smaller pipette because there is more room for error. Because of this, when pipetting more than 10 uL, do it in increments. (Eject two sets of 5.25 uL for 10.5 uL of media).
11. After calculating, add the specified amount of water and primer into the small test tube as well with a micropipette, changing tips each time you pipette. Label this as “Test” and be sure to initial the tube with the groups initials to avoid confusion.
12. Next, we will create the control tube. With the other small tube full of 12.5 uL of master mix, add 1 uL of primer and 11.5 uL of water, without pDNA. This is to ensure that the primer and master mix aren’t contaminated, as this could happen by human contact in lab or during creation.
13. Make a chart of this data for later reference, including sample name and amounts of 2x TAQ mix, pDNA, primer, and water for a total volume of 25 uL .
14. Place the control and test tubes in the tube holder at the front of the classroom filled with ice. Be sure to label your caps with sharpie, and record on the spreadsheet which wells are yours.
15. Next, we will review for the midterm using Kahoot.
16. Afterwards, make sure that your lab benches are clean, and complete and turn in the QTM. Review for the midterm next week.

Data and Observations:

Pictured below is the ice basin with the pDNA, control, test, dilution, and primer.

Below is the picture of the chart used for identifying which spot the control and test tubes were placed in.

Picture below is the tube holder with each group’s test and control. As seen, our group had used wells C3 and C4.

Below is a chart of the tube contents for both trials:

Storage:

The ice tray was placed at the front of lab, and the control and test tubes were placed into the tube holder. The pDNA, water, and primers were placed back into the cooler. The benches were wiped down and micropipettes were placed back on the rack.

Conclusions:

In conclusion, our group worked together very well, and tested the scientific process as we proceeded. Although some mistakes were made, we corrected them and fixed problems as they came along. We were also very good at maintaining an aseptic environment, as we would close caps and micropipette tip boxes when not in use. Overall, we learned a great deal about polymerase chain reactions, and the man that discovered it (while supposedly on drugs). We also learned about the three processes of PCR, denaturing, annealing, and elongation, and practiced our knowledge with Kahoot. Overall, we set up samples for next lab to begin PCR analysis, and started to think about the poster projects in the future.

Future Steps:

In the future, I would have made sure the group used a p10 pipette through the duration of lab, due to the fact that we had to start over the control because a larger micropipette was being used. If not noticed, this could have ruined our data, as a larger pipette isn’t as accurate when it comes to measuring very small amounts.

February 28, 2019

Rithvik Baratam

I. Title: Polymerase Chain Reaction of pDNA

II. Rationale:

The rationale behind this lab was to learn how to use PCR to amplify the pDNA from the previous lab. It is an easy way for us to study targeted areas of genes. PCR can help us in the identification and classification of our soil ciliates.

III. Materials:

• Bleach
• Gloves
• 10µl micropipettes
• Taq polymerase
• DI water
• Primer
• Nucleotides

IV. Procedure:

Creating a Sterile Environment

1. Ensure that gloves are worn and are on throughout the experiment.
2. Wipe down work station with bleach to disinfect the working area.
3. Minimize the amount of movement and air flow in the work station.

Preparing DNA

1. Make sure that the DNA is approximately 100 ng for each sample
2. If the concentration of DNA is over 1oo ng/µl dilute the DNA
3. Our concentration of DNA was 1115 ng/µl and was diluted in a 1:10 dilution. This was done by taking 1 µl of 1115 ng/µl DNA and adding 9 µl of DNAse free water.

Making a Control Sample

1. 12.5 µl of Taq polymerase was added to a tube and placed in an ice bath
2. 1 µl of primer mixture was added to the same tube
3. 11.5 µl of DNAse water was added to the same tube, adding up to 25 µl in total

Making a Treatment Sample

1. 12.5 µl of Taq polymerase was added to a tube and placed in an ice bath
2. 1 µl of primer mixture was added to the same tube
3. 1 µl of diluted 111.5 ng/µl DNA was added to the same tube
4. 10.5 µl of DNAse water was added to the same tube, adding up to 25 µl in total

V. Conclusion:

The control and treatment were labeled and stored in the ice bath so that it would incubate. These tubes will then be placed in a thermocycler for the reaction to begin. We will then run an agarose gel electrophoresis to measure the DNA segments against a DNA ladder. The experiment taught us how to set up a polymerase chain reaction of the DNA that we purified. It broadened our scope of knowledge regarding DNA analysis. In the future, I hope to continue to learn more about at thermocycler and eventually metabarcode our DNA.