lab 7: pre-PCR
Lab seven: pre-PCR
Adriana Robledo
2-28-19
purpose:
The purpose of this lab was to practice dilutions through working with our templates in order to prepare for the amplification of our DNA.
PCR assay
- Bleach the work table and any tools used.
- Obtain two centrifuge tubes (one control and one treatment) with 12.5 μl of Master mix and 1 μl of primers already in either tube.
- Seeing as the maximum amount of DNA that can be added is 5μl, a dilution needs to be made using the concentration food using the nano drop in the last lab.
- Once the amount of μl of DNA and water required for dilution have been found (keeping in mind that the total volume equal and not exceed 25μl), add the amounts to the tube containing the “Taq Mix”
- Add the given amount of water the the control tube, and centrifuge them both for equal mixing.
- Label the control and treatment groups with the group’s initials, place them in the rack to be put through the thermocycler and write down and record the row and spot you placed the samples.
results:
| Tube | 1 (Euk) | 3 (Control Euk) |
| 2X Taq Mix (μl) | 12.5 | 12.5 |
| DNA (μl) | 3.8 | 0 |
| 10 μl Euk Primers (μl) | 1 | 1 |
| Water (μl) | 7.7 | 11.5 |
| Total volume (μl) | 25 | 25 |
DNA concentration: 262.1 ng/μl
1:10 dilution
262.q/10 = 26.2
100/26.2 = 3.81 μl
conclusion:
Our lab group was able to put together all of the correct components for the setup of PCR. Our calculations were double-checked and correct so we were able to move forward very quickly upon completion of the math. Our knowledge was tested and secured with a Kahoot which asked about the article “Multiple marker parallel tag environmental DNA sequencing reveals a highly complex eukaryotic community in marine anoxic water,” an article that we have been looking at for weeks. The Kahoot also asked questions about concepts we have been working with in class, and offered Dr.Adair an opportunity to make sure that we are all on the same page. We completed our QTMs that had us predict the next steps for our lab, which we concluded to be PCR and even gel electrophoresis to test our success with DNA extraction. Lastly, we planned out the format of our poster that we will present after the completion of this experiment.
Future steps:
In the next lab, we plan on finishing the process (autoclaving, annealing, and expanding) for PCR. I hope to work as quickly and efficiently as we did in this lab, and hopefully be able to keep the temperatures consistent with where they need to be.
Storage:
Our DNA was stored in A5 and A6 on the rack. The pipet tips were disposed of and any excess DNA was refrigerated.