February 22, 2018

Purpose:

The purpose of this lab was to learn about the process of PCR and carry out the beginning of the protocol with our soil DNA samples and a positive and negative control. Students are also to make agarose gel and construct the gel electrophoresis apparatus for later use.

Procedures:

Part 1:

1. Discuss learning objectives with class.
2. Draw out process diagram for PCR.

Part 2:

1. Prepare lab for PCR by wiping down the lab tables with bleach, tying back hair, and putting on gloves.
2. Obtain 3 tubes of pre-assorted Master Mix (12.5 uL in each)
3. Label each tube on the side with a sharpie (+,-, S)
4. To the negative tube, using a p10 micropipette add 1 uL of the primers (COX 1 forward and reverse), and 11.5 uL of water (6.5 uL first followed by 5 uL for a total 11.5 uL).
5. To the positive tube, using a p10 micropipette add 1 uL of the primers(COX 1 forward and reverse), 6.50 uL of distilled water, and 5 uL of the control DNA.
6. To the tube labeled S, using a p10 micropipette add 1 uL of the primers(COX 1 forward and reverse), 6.50uL of distilled water, and 5 uL of the soil DNA.
7. Label your group number on the top of each tube with a sharpie.
8. Place the 3 tubes in the tube rack at the front of the classroom making note of the location you put it in. Record number/letter placement label on QTM sheet.

Part 3:

1. Using analytical balance, weigh out 0.6 grams of agarose, and add it to Erlenmeyer Flask.
2. Add 40mL of TAE (accurately measured with a graduated cylinder)  to the flask.
3. Close the flask with paper and stopper.
4. Microwave it for 1 minute and 20 seconds at a power of 7 making sure it is boiling.
5. Place the flask in a 55 degrees Celsius water bath for 5 minutes.
6. Add 2uL of ethidium bromide and mix.
7. Assemble electrophoresis apparatus, making sure there are no openings and pieces are compacted.
8. Slowly add the gel to the apparatus, and allow it to cool for 20 minutes before storing in the freezer.

Data:

The initial concentration of the stock primer was 10 uM.

The final concentration in our sample was 0.4 uM.

Tubes placed in A1, A2, A3.

Conclusion:

This lab was successful as we were able to prepare our trial samples for PCR. Our results from last week did not come out the way we had hoped (NanoDrop curve was significantly off) so we were unable to get an estimate at the concentration of DNA we had in our sample. Most of the components were already pre-assorted by the lab team, so it was easy because we were just adding things to the mix. The most difficult part of the lab was making sure that we were keeping a sterile environment. We did this by wearing gloves, cleaning out tables with bleach, making sure we weren’t speaking in the direction of the samples, and making sure the pipette boxes and tubes were being closed. The next step is to run our trial samples under current in gel electrophoresis to make sure we get bands. We will then send our samples to a lab for analysis and sequencing. In the future, it will be beneficial to have half the class doing the protocol in a slightly different way so we have a better chance of getting positive results.

Gel Apparatus labeled: Group 7 & Stored: in freezer