Lab #7: PCR Amplification of DNA
Lab #7: PCR Amplification of DNA – February 28th, 2019
Objective
For this weeks lab, we are continuing our analysis of the chelex purified DNA samples that have been collected from the soil ciliate cultures by perming a PCR (Polymerase Chain Reaction). Now that we have confirmed that there is DNA in the sample through the gel electrophoresis and through the nanodrop spectrophotometry assays, we are ready to select a specific gene within our sample and amplify that gene through a PCR.
Rationale
We are analyzing our DNA sample through a PCR in order to address the experimental question: What is the biodiversity of Baylor trees? A PCR will allow us to target a specific gene that is not conserved so that we can send the DNA to a lab for analysis.
Methods
PCR – Loading the tube
12.5 microliters of 2x Master Mix were placed into 2 autoclave tubes, one serving as the control which will not have any DNA sample in it. The master mix contains DNA polymerase, free nucleotides, and buffer. WIth a nanogram/microliter concentration of 112.1, only 1 microliter of sample DNA was needed in order to have enough DNA. The 1 microliter of DNA sample was placed into the non-control tube. 1 microliter of 10 micromolar 18sv4 primer was placed into both the control and non-control tubes. 10.5 microliters of nuclease-free water were added to the non-control tube and 11.5 microliters of nuclease-free water were added to the control tube. The tubes were placed in a rack at the front of the room. The tubes will be placed into a thermocycler to perform the PCR, and the amplified DNA will be sent to a lab for analysis.
Amounts used for PCR:
| Tube | DNA Sample | Control |
| 2X Master Mix (µL) | 12.5 | 12.5 |
| DNA (µL) | 1 | 0 |
| 10 µM 18SV4 primers (µL) | 1 | 1 |
| Nuclease-Free Water (µL) | 10.5 | 11.5 |
| Total Volume | 25 | 25 |
Results
The procedure was completely finished, but unfortunately, we ran out of time to see the end result of the PCR so the results will be observed next week. The lab results will most likely be ready after a few weeks.
Conclusion
The PCR procedure was very easy to follow, as it really only involved minor calculations on how much DNA sample to add to the tube. Since I know that my sample had DNA according to the nanodrop assay and gel assay, I am fairly certain that the results from the PCR will be useful for our experimental question.
Future Goals
After the PCR runs, the samples will be sent to a lab for analysis and the information about our soil ciliates will be able to be analyzed. We plan on designing a poster for the symposium that is coming up, which will include everything relating to this experimental question.
Storage
The samples ready for PCR were left on a rack, they will go through the thermocycler next and stored in a fridge before being sent off.