Purpose:

The purpose of this lab was to analyze the results of our PCR from our tubes that we produced in an earlier lab. We ran our positive, negative, and environmental DNA samples through gel electrophoresis to determine if DNA was present in our amplified samples to see if our DNA extraction methods were successful or to determine if we needed to try a new extraction procedure if ours was unable to produce results.

Procedure:

• Gel electrophoresis:

1. Prepare 300 mL of 1X buffer solution using 10x TAE stock solution and D.I. water. Do this by measuring out 270 mL of D.I. water in a large graduated cylinder and adding it to a 500 mL erlenmeyer flask. Pipette 30 mL of 10x TAE stock solution and add to the flask using a serological pipette.
2. Place the agarose gel into the gel electrophoresis apparatus, with the wells closest to the negatively charged side of the box.
3. Pour in the 1x buffer solution onto the gel, filling the box and covering the gel and the wells completely.
4. Have each group member practice loading a well with a pipette, using whatever leftover diluted buffer as your sample.
5.  Collect your positive and negative control samples from the front of the room. Carefully micropipette 10 µl of each sample into separate wells in your gel.
6. Micropipette 5 µl of DNA ladder already mixed with loading dye into the well adjacent to your samples.
7. Connect the positively and negatively charged wires to their appropriate place on the box and to the power supply.
8. Place the lid onto the apparatus and turn on the power supply to the wires. Run the gel electrophoresis for about 30 minutes at 100 V.
9. Remove the gel after 30 mins and analyze under UV light to examine the bands of DNA present in the gel.

Data:

***Our wells were labeled 1-6, running from the right side of the gel to the left side. Well 1 begins on the right with our DNA ladder.

Well #:

1. DNA ladder
2. Negative control (COX 1 primers)
3. Positive control (COX 1 primers)
4. Environmental DNA (COX 1 primers)
5. Negative control (V4 primers)
6. Positive control (V4 primers)
7. Environmental DNA (V4 primers)

Conclusion:

Our group’s gel electrophoresis only showed results for the DNA ladder and the positive control using COX 1 primers. The rest of our wells showed no results. This could either be because the COX 1 primers were better primers to use for our experiment than V4, our DNA extraction method was not consistent, or our PCR protocol was contaminated or had something that prevented the DNA from being amplified properly. We will have to try our DNA extraction procedure again, or perhaps try the Chelex protocol instead to see if we obtain any better results. There could be a number of reasons that our gel electrophoresis showed that we didn’t have DNA in any of our other wells, however we will have to go through our procedure again to test new changes or try different techniques to see if they will prove to be more successful for our protocol in the future. Our tubes were stored in the box with everyone else’s PCR samples at the front of the room and are labeled Group 7. Our gel was disposed of after we had finished photographing and analyzing it.