Lab 7: PCR Amplification of DNA
Daphne Simo
03/01/19
I. TITLE
PCR Amplification of DNA
II. RATIONALE/PURPOSE
The purpose of this lab was to perform a polymerase chain reaction on our chelex DNA or eDNA.
III. MATERIALS
Purified DNA sample
Taq mix
Euk Primer
Micropipettor
Water
Microfuge tubes
Centrifuge
Ice
Small container
IV. PROCEDURE
Aseptic Preparation
- Wear gloves.
- Clean lab table with 10% bleach.
Polymerase Chain Reaction procedure
- Collect tubes of primer, purified DNA, water, and taq mix and place into a small container with ice.
- Obtaining one microfuge tube, place 12.5μl of 2x taq mix, 2μl of purified DNA, 1μl of 10μM primer, and 10.5μl of water into it.
- Obtaining a new microfuge tube, place 12.5μl of 2x taq mix, , 1μl of 10μM primer, and 12.5μl of water into it.
- Label the first microfuge tube as treatment and the other as control.
- Place the two microfuge tubes into a centrifuge for about 1 minute, while balanced, to make sure all the components are mixed well.
- Place the control and treatment tubes into specified wells.
V. DATA/OBSERVATIONS
| Tube | 1 (Control) | 2 (Extracted DNA) |
| 2x Taq Mix | 12.5 | 12.5 |
| DNA (μl) | 0 | 2 |
| 10 μM primers (μl) | 1 | 1 |
| Water (μl) | 11.5 | 10.5 |
| Total Volume (μl) | 25 | 25 |
VI. STORAGE
The water, primers, 2x taq mix, and DNA were placed back into the coolers. The microfuge tubes with and without the DNA were placed into c5 and c6 wells accordingly.
VII. CONCLUSION
This was my first encounter with polymerase chain reaction in terms of learning what it is and actually performing one. The process was very straightforward, however there was a bit of math involved trying to make sure the concentration of the purified DNA matched with the other components (primer, taq mix, etc.). This was due to the fact that our purified DNA was around 244ng/μl, which was close to the goal of 100ng/μl, so if we were to perform a 1:10 dilution, our DNA concentration would be lower. Instead, we chose to do a 1:1 dilution, and it worked out well.
VIII. FUTURE STEPS
Moving forward, I hope to see good results after we denature the DNA. I want to learn more about how the DNA anneals, as the process confused me a bit. Furthermore, I hope to have a good grasps on the concepts we learned this semester so I can do well on the midterm quiz next time we are in lab.