Purpose: Prepare negative and positive samples as well as the soil DNA samples in order to go through PCR amplification. Also, prepare the agarose gel and wells in order to conduct gel electrophoresis.

Procedure

-Preparing the Controls and Soil Sample

1. All students must be wearing gloves and be careful not to contaminate the samples
2. Clean the work area using bleach
3. Label the 3 different tubes containing 12.5 uL of 2X master mix with + (positive control) , – (negative control) and S (soil sample)
4. Add the appropriate amount of culture, primers and water to each tube
5. Mixtures
   1. Positive Control: 1 uL of Cox1 primers/ 5 ul of 10ng/ul culture/ 6.5 ul of deionized water
   2. Negative Control: 1 ul of Cox1 primers/ 11.5 ul of deionized water
   3. Soil Sample: 1 ul of Cox1 primers/ 5 uL of eDNA smaple/ 6.4 ul of deionized water
6. Micropipette each solution using a different tip
7. place all three tubes in a tray with each tube labled with group number

-Preparing Agarose Gel

1. Add 40 mL of 1x TAE to 0.6g agarose in a small Erlenmeyer flask
2. Mix it for a bit and put in the microwave (put the weigh paper on top then loosely put the cap on the flask)
3. Microwave the flask for 1 minute and 20 seconds under power 7
4. Gently swirl the sample and place it in the water bath for 5 minutes
5. While the sample is being cooled, set up the gel electrophoresis box, making sure the open ends are sealed
6. After the flask is cooled, add 2uL of ethidium bromide (Done by the instructor)
7. Swirl the mixture and gently pour it into the prepared mold.
8. Allow it to solidify for a few minutes
9. Cover the gel with prepared buffer solution so it would not dry out
10. Label box and turn it into the L.A.

Results

Each tube labeled +,-, S. All were labeled with #3 and stored in the tray.

Gel electrophoresis box was labeled JJN #3 and turned it into L.A.

Gel electrophoresis would be conducted in next week’s lab

Conclusion

It was interesting to see the actual process of creating the agarose gel. I am excited to actually use the DNA samples and conduct gel electrophoresis for each sample that is made. Also, it was interesting to see each process in detail and learn what each chemical does. For example, the primers are there to aid in the process of PCR amplification and allow DNA polymerase to bind to the correct place in the DNA. Also, ethidium bromide is added to allow to visualize the movement of DNA across the gel.