3/22/18

Purpose: The purpose of today’s lab was to continue with the protocol we did last week for DNA extraction and take on the next step in research: PCR. We used the V4 and COX 1 primers to prep our PCR so next week we can perform gel electrophoresis.

Procedure:

A. PCR

1. Use the table in the QTM worksheet to determine how much DNA template, 2X master mix, COX1 primers, and water is needed for our negative control (-), positive control (+), and environmental DNA (e).
2. Obtain 6 microcentrifuge tubes with mastermix from Dr. Adair.
3. Label each tubes with their appropriate label: 1+ LAK, 2- LAK, 3e LAK, 4+ LAK, 5- LAK, 6e LAK.
4. Add the appropriate, calculated amounts of template, master mix, primers, and water to each microcentrifuge tube.

B. Agarose set-up

1. Weigh out .6 g of agarose on a weigh paper and place it into a flask.
2. Add 35 ml 1xTAE to the flask.( Our TAE was made by adding 90 mL of DI water to 10 mL of 1X in a flask while swirling.)
3. Cover the flask with the weighing paper and loosely place a cap on it.
4. Heat solution in the microwave for 2 minutes at power 7 until the solution is clear.
5. Allow the solution to cool.
6. Have Dr. Adair add 2 ul ethidium bromide and swirl.
7. Assemble the electrophoresis box.
8. Pour agarose smoothly into prepared mold to avoid creating bubbles.
9. Allow agarose to sit and harden.

Data:

Above is our 6 labeled microcentrifuge tubes.

These are the 5 tubes we obtained from Dr. Adair. It contains our control and study, the primers, and water.

Above is a picture of the .6 Agarose we weighted out and the solution immediately after we swirled it together.

This is our electrophoresis box assembled with my groups identification label on it.

Future Studies: The next step after today’s lab would be to run PCR and then proceed to performing gel electrophoresis. Fingers crossed that we did everything right today and our PCR is successful so we can come back next week and perform gel electrophoresis and study our gel under the light to see if we have a matching genetic barcode.

Conclusion: It’s so cool to look back on this semester and see how far we’ve come along and how much we’ve learned. Learning diligence isn’t something I thought I’d learn in bio lab, but it’s actually one of the most important things I have learned. I’m glad our numbers showed that we did have DNA. I think it was super comforting for my group since the last time we got our numbers back from the nanodrop, our DNA concentration was almost 0. I’m excited to continue with our protocol and hopefully write a paper about its success.

Storage: All of our tubes were stored in the blue tube rack along with the rest of the class`s tubes; ours specifically had LAK on the top of them. Our agarose gel, labeled LAK 21, was on our table until the next lab came in and after it will be stored in a freezer.