February 28, 2019

Noah Mendoza

Purpose:

Preform a PCR to amplify the purified DNA made during previous labs, using primer sequences. The results of our PCR will help us to identify the organisms, ciliates, found in our soil samples.

Materials:

1. Bleach
2. Gloves
3. 0.5-10 μL Micropipette w/ tips
4. Autoclaved D.I. water
5. Taq Polymerase
6. DNA Primers
7. Nucleotides

Procedure:

Creating a sterile Environment and working Aseptically

1. Put on gloves and be sure to wear them over the duration of the experiment. Change gloves if necessary while carrying out the procedures for the experiment.
2. Spray a bleach solution onto the work bench and thoroughly wipe down the entire working area.
3. Minimize the movement around the work bench and minimize the possibility of contamination as much as possible.

Preparing the DNA

1. Dilute the DNA sample to ensure that 100 ng of DNA is present in each sample.

• Our DNA sample contained 1115.16 ng per μL
• Take 1 μL of DNA sample and place it into a separate test tube to make the dilution
• Add 9 μL of auto calved D.I. water, this makes a 1:10 dilution ratio
• We than took 1 μL of the diluted DNA, which contained 100 ng of our pDNA

Making a Treatment Sample

1. 12.5 μL of Taq polymerase was added to a test tube and placed into an ice bath and given to us
2. 1 μL of primer was added to the test tube
3. 10.5 μL of autoclaved D.I. water to the test tube
4. Add 1 μL of the diluted DNA sample to the test tube

Making a Control Sample

1. 12.5 μL of Taq polymerase was added to a test tube and placed into an ice bath and given to us
2. 1 μL of primer was added to the test tube
3. 11.5 μL of autoclaved D.I. water test tube

Conclusion:

The control and treatment were labeled and placed into the ice bath in spots C 11/12. The test tubes will be running through a thermocycler at different temperatures for different time intervals in preparation for next week’s lab, where we will run our PCR against a DNA ladder in an agarose gel.

Future Goals:

In the future, we will get our results back from our PCR primer sequences and be able to predict what organisms are present from our eDNA samples. We will also begin work on our presentation posters, having completed a rough draft and setting up a plan for completion in today’s lab.