Lab#7
Purpose:
The purpose of this lab was to learn about the V4 ribosomal primer, PCR, and scientific poster designs. We set up the purified DNA sample for PCR so it can be put into a thermal cycle to make more copies of out targeted DNA. The thermal cycle will be used to make more DNA in a three step process, denature, annealing, and elongation.
Material:
- Cleaning supplies
- Micropipette
- Clean tubes
- V4 primer
- Master Mix
- Sterile water
- Small ice tub
Procedure:
- Clean the lab table with 10% bleach solution to prevent the sample from being contaminated for the PCR. Clean pipettes as well and wear gloves.
- Place water, primer, DNA sample, 2x taq solution, and microtube in a small ice container.
- Calculate the amount of DNA and water that will be added to the tube already containing 12.5μl of 2x taq mix.
- Perform a dilution of the DNA if there is a large amount of DNA. Start by placing 9μl of water and 1μl of DNA in a clean tube and mix by pipette. Centrifuge if necessary to bring all the solution to the bottom of the tube.
- Pipet 2μl of diluted sample into the tube already containing 12.5μl of 2x taq mix.
- Pipet 1μl of primers into the tube already containing the 2x taq mix.
- Pipet 9.5μl of water into the same tube and label it as Soil DNA.
- Repeat in a control tube with all components except DNA sample and will receive 11.5μl of water.
Data:
| Control | Soil DNA | |
| Master Mix | 12.5μl | 12.5μl |
| DNA Sample | 0 | 2μl |
| Primers (10μl) | 1μl | 1μl |
| Water | 11.5μl | 9.5μl |
| Total Volume | 25μl | 25μl |
Storage:
The tubes containing primers, DNA sample, and water were stored in a cooler. The tubes containing the remainder from the diluted DNA sample was disposed of properly.
Conclusion:
During this lab we were able to prepare our DNA extracted samples for PCR. We will then place our samples in an ice box so they can be taken to do thermal cycling later. In the thermal cycle process, the DNA will be denatured, annealed, and elongated to make more copies of the DNA. After this we will be able to sequence and analyze the DNA to gain further knowledge into the organism that live in the soil ecosystem and hopefully be able to identify and characterize more ciliates.
Future Goals:
In future labs I would like to use the DNA that went through PCR and run it in Gel Electrophoresis. Using Gel Electrophoresis we will be able to see if the PCR was successful and to see if the DNA can be used in future experiments. I am hoping that we are going to be able to analyze the DNA to identify and characterize more ciliates.