Lab #7: PCR Amplification
Michelle Nguyen
2/28/19
Lab Notebook #7: PCR Amplification
Objective/Purpose: The objective of this lab was to perform PCR and to begin designing our scientific posters.
Procedure:
PCR
- Bleach the tables (surrounding area) to avoid contamination of samples.
- Calculate the amount of DNA and water that is needed to perform PCR and set up your treatment tube (containing extracted DNA).
- After making the calculations, perform a dilution (as necessary) by adding 9 μl of water to a sterilized tube and 1 μl of extracted DNA and mixing the solution via pipette. Centrifuge as necessary to ensure that solution is all at the bottom of the tune.
- Obtain a separate tube already containing 12.5 μl of 2X Taq Mix. Label each tube control or treatment.
- To the control tube, add 5 μl of water and 1 μl of 10 μM primer. Mix via pipette.
- To the treatment tube, add 1.7 μl of DNA sample, 0.4 μl of 10 μM primer, and 9.8 μl of water. Mix via pipette.
Data
(600 ng/μl)(x μl) = 100 ng
X = 0.17 μl
1:10 DILUTION
(60.0 ng/μl)(1.7 μl) = 100 ng
Water (μl) added: 25 – (12.5 + 1.7 + 1)μl= 9.8 μl
| Tube | 1 (Extracted DNA) | 2 (Control) |
| 2x Taq Mix | 12.5 | 12.5 |
| DNA (μl) | 1.7 | 0 |
| 10 μM primers (μl) | 1 | 1 |
| Water (μl) | 9.8 | 11.5 |
| Total Volume (μl) | 25 | 25 |
Conclusion: During this lab we were able to prepare our DNA extracted samples for PCR in the following lab. Through the amplification of our DNA samples, we will be able to sequence and analyze them, gain further insight into the organisms that live in the soil ecosystem of our tree, and hopefully be able to identify and characterize ciliates.
Storage: The tubes containing primers, our DNA samples, and water were re-stored in a cooler. The tube containing the remainder of the diluted DNA sample was disposed of.
Future Steps: We will perform PCR and gel electrophoresis once again.