Date of Experiment: February 28, 2019

Objectives:

• Antiseptic techniques
• Develop control and experimental PCR samples
• Outline group presentations

Purpose: Setting up a PCR reaction of the chelex DNA, and sequencing the amplified DNA to observe ciliate biodiversity

Materials:

• Gloves
• 10% bleach
• Pippettes
• Test tubes
• Ice tray
• Water
• DNA sample (uL)
• 10 uM Euk primer (uL)
• 2X Taq Mix (uL)

Methods:

1. Clean the area with 20% bleach, using gloves before and throughout the experiment
2. Place 12.5 uL of 2x Taq Mix in two separate test tubes and suspend these in ice (these will serve as the control and experimental sample)
3. To reach 25 uL for the control sample, place 2.5 uL of primer and 10 uL of water in one of the 2x Taq Mix test tubes
4. To reach 25 uL for the experimental sample, place 3.5 uL of DNA sample, 2.5 uL of primer, and 6.5 uL of water in the remaining 2x Taq Mix test tube
5. The polymerase chain reaction will begin in the thermocycler as the DNA is denatured, annealed, and elongated

Observations:

The Nanodrop results from the previous lab displayed that the DNA concentration was about 32 ng/uL. Upon gel electrophoresis of the PCR samples concentrations, it will be observed whether DNA  amplification was significant to that of the initial concentration.

Results:

The PCR control sample required: 12.5 uL 2x Taq Mix, 0 uL of DNA sample, 2.5 uL of 10 uM Euk primer, and 10 uL of water.

The PCR experimental sample required: 12.5 uL of 2x Taq Mix, 3.5 uL of DNA sample, 2.5 of 10 uM Euk primer, and 6.5 uL of water

Storage:

The laboratory area was cleaned with 10% bleach, gloves and micro-pipette tips were disposed, PCR samples were labeled and stored in a cooler alongside the primers and H20 test tubes. The ice trays were disposed of in the sink and dried.

Conclusion/Discussion:

Upon PCR, or polymerase chain reaction, of a specific DNA sequence, gel electrophoresis will be needed to observe whether much of the DNA was amplified. If amplified, these findings will then be sequenced for comparison to that of known ciliate DNA and those of other DNA samples within the lab. Such findings not only give insight to ciliate biodiversity among Baylor soil, but also how these genetic differences allow for ciliates to function effectively.