Task, Rationale, and Purpose: Lab week 12 was a repetition of the Chelex protocol with some modified components such as the primer concentration used. The task of the lab was to successfully repeat the Chelex protocol, analyze the eDNA sample using a nanodrop, and prepare the agarose gel for gel electrophoresis. The purpose or rationale of the lab was to test the ability of the Chelex protocol to provide purified DNA and, if sufficient, sequence the DNA in later experiments to determine the diversity of the organisms in our Bermuda grass soil.

Modified Chelex Protocol:

1. Using a micropipette p1000, add 1.5 µl of bermudagrass soil non-flooded plate water to a microcentrifuge tube.
2. Label the tube with the group’s number.
3. Centrifuge the tube for 5 minutes at 6000 x g.
4. Remove the supernatant.
5. From the non-flooded plate, take 1.5 mL of liquid and add it to the microcentrifuge tube.
6. Centrifuge the tube for 5 minutes at 6000 x g.
7. Remove the supernatant.
8. Repeat steps 1-7 two times.
9. Add 200 µl 5% Chelex to each tube, and vortex for 1 minute.
10. Using a large-bore micropipette tip, add 15 µl of proteinase K to each tube.
11. Incubate the tubes for 30 minutes in 56^oC water bath or heat block.
12. Boil the tubes for 8 minutes in 100^oC water bath or heat block.
13. Vortex the tubes for 1 minute.
14. Centrifuge the tubes at 16,000xg for 3 minutes to pellet cellular debris and Chelex beads
15. Transfer 100 µl of supernatant from each tube into a clean microcentrifuge tube.
16. Label the top and the side of the microcentrifuge tube with the group initials, section and group number, chDNA, and the date.

Nanodrop Procedure:

1. Using a micropipette p10, add 10 µl of DI water to the nanodrop.
2. Calibrate the nanodrop.
3. Clean the DI water off of the nanodrop and add 10 µl of the supernatant obtained from the Chelex protocol.
4. Use the nanodrop to analyze the DNA concentration, its A260/280 ratio, and A260/230 ratio.
5. Dilute 1 µl of the supernatant to obtain a DNA concentration that is approximately 50 ng/µl.

PCR and DNA Tube Procedure:

1. Ensure that the lab table is bleached and all participants are wearing gloves.
2. Obtain three microfuge tubes containing 12.5 microliters of 2X master mix.
3. Label one tube “22-8+”, the second tube “22-8 -“, and the third tube “22-8 e”.
4. Using a micropipette p10, add 1.25 microliters of 10 µM stock PCR V4 primers to each tube.
5. Using a micropipette p10 with a new tip, add 1 microliter of the paramecium control obtained from lab week 10 into the + tube.
6. Using a micropipette p10, add 1 µl of eDNA supernatant (50 ng/µl) obtained from the Chelex protocol to the e tube.
7. Using a micropipette p20, add 11.25 microliters of DI water to the – tube.
8. Using a micropipette p20 with a new tip, add 10.25 microliters of DI water to the + tube.
9. Using a micropipette p20 with a new tip, add 10.25 microliters of DI water to the e tube.
10. Place the tubes in the thermocycler and record their location and designate them with a group number and group initials.

Agarose Gel Protocol:

1. Using a graduated cylinder, measure out 35 mL of 1xTAE.
2. Add 0.6 grams of agarose to an Erlenmeyer flask along with the 35 mL of 1XTAE solution.
3. Cover the flask with a Kimwipe and a cap. Ensure the cap is loose.
4. Heat the solution for 1 minute and 20 seconds.
5. Cool the gel for 5-6 minutes in a water bath.
6. While cooling, set up a gel electrophoresis box.
7. Add 2 microliters of ethidium bromide and swirl.
8. Pour the cool agarose gel into the mold and use a micropipette to pop any bubbles that form.
9. Insert the comb into the furthest holder to the back of the box.
10. Label the gel electrophoresis box with a group name, number, and teacher.
11. Give the box to the TAs for storage.

Nanodrop Data:

ng/µl: 365.2

A260/280: 1.31

A260/230: 0.51

Diluted using a 5 µl supernatant to 45 µl DI water concentration

Thermocycler and Agarose Gel Storage: The environmental, positive, and negative control samples were placed in B2-10, B2-11, and B2-12 respectively. The gels and the dilution tubes were given to the TAs for storage.

Conclusion: The lab took a while to complete due to the mistake of following last week’s PCR and DNA Tube Procedure which had to be repeated. The chelex protocol resulted with two tubes containing dark supernatant and one tube containing a clear supernatant. The dark tubes most likely had a lot of debris from the soil still contained in it. This is what most likely led to a high ng/µl rating. For future experimentation, the supernatants from each group will be pooled. One metabarcoding reaction from the pooled positive PCR reactions will occur.