Holli Brown

CILI CURE 21

2/21/19

Objectives:

The objectives for this lab were for students to successfully complete a gel electrophoresis test, as well as analyze their DNA for purity. Students will correctly assemble the electrophoresis apparatus, as well as practice pipetting into the gel wells, as this process is used in DNA analysis many times. Students will also learn how to prepare the DNA samples, and understand why the wells are placed by the cathode rather than the anode. Students will also review for their midterm in class, and solve problems as they arise. During gel and nano drop analysis, students will understand why results happened the way they did, as well as understand the processes behind each test. Lastly, students will compare their nano drop DNA tests to ideal tests, as well as analyze their gels under a gel imager machine. Students will take good notes throughout the process, and use critical thinking skills and aseptic techniques throughout lab.

Methods and Lab Summary:

1. Obtain the agarose gel made last lab, however we will only need one per table. It will be helpful to draw out a ‘map’ of your agarose gel, in order to remember the contents within each lane.
2. Each group will need a mass standard, one high and one low. The high should be from 250-500 ng, while the low should be around 15-125 ng.
3. Label your map of the wells, and include which medias went where. When analyzing the gel, it will be much easier to compare results if the contents of the well are known.
4. In order to make the DNA sample, pipette 9 uL of  DNA media from the Eppendorf tube made last lab into a very small eppendorf tube, and mix it with 1 uL of loading dye. This loading dye will help the media to fall into the well because it is dense, as well as aid in visualizing the progress of electrophoresis so the DNA doesn’t run off the gel.
5. Before ejecting the medias into the well, make sure you are practiced. Use the practice well and media at the front of the lab, and try injecting into the well. When you have been successful, move onto the real proccess.
6. After this practice, pipette the 10 uL of DNA media into a lane. Make sure you label where this goes, and record it in your notebook.
7. Choose 2-3 mass standards, and pipette these into the wells. Label which mass standards went in each lane, as we will use these to compare the DNA samples to.
8. After the well has been filled by both your group and your partner group, place it into the electrophoresis apparatus, filled with buffer.
9. Take the black rubber holders and comb off the gel mold, so that the clear plastic part and the gel remain.
10. Place the gel with the wells closest to the black part of the tank, as this is the negatively charged cathode. The anode is the red side, which is positively charged.
11. Attach the cables to the power source, the red cable goes into the red plug and the black cable goes into the black plug.
12. Turn the machine on, and select the constant voltage option. This will allow a constant voltage to disperse across the gel, allowing for slower migration, and it allows for more diffusion.
13. Let the machine run for 20-30 minutes. The machine is working properly when bubbles can be seen forming at the cathode and anode ends.
14. Play the Review Kahoot while waiting, and make sure you review for the midterm for Week 8.
15. After 20 minutes have passed, remove the gel from the electrophoresis machine and transfer it into a plastic tub.
16. We will now go upstairs to lab C305 and analyze the gel and DNA samples.
17. With Dr. Adair, place the gel into the gel imager machine, being sure to wipe down the glass surface first. Slide the apparatus back into the machine, and hit start.
18. This machine will analyze the gel, and produce a black and white image for us to see. It uses blacklight so that the Ethidium Bromide will be visible, and takes a picture that can be uploaded to the computer.
19. Look at the image. Is it closer to the low or high mass standards? Can you see DNA?
20. Afterwards, move to the back of the lab with Mike. Here, we will use the Nanodrop One machine to analyze the DNA samples in the eppendorf tubes. This machine uses spectrophotometry to measure DNA purity.
21. Clean the upper and lower optical lenses, in order to prevent contamination of your sample.
22. First, we will blank the machine with 2 uL of deionized water, as this will allow the machine to form a blanked base measurement.
23. Afterwards, wipe the surface down with a KimWipe, and pipette 2 uL of DNA sample onto the optical surface. This larger drop will ensure the liquid doesn’t slip off the optical lens, and that it is centered.
24. Close the pedestal, and obtain a reading. This information can be compared to other groups, as well as other experimental samples.
25. Return down to the lab, and finish your QTM. Be sure your lab space is cleaned, and be sure to place the gel back into the fridge.

Data and Observations:

The media below is the mass standards of 250 and 500 that were used in gel electrophoresis.

Below is the gel electrophoresis device, with the black end being negatively charged and the red end being positively charged. The DNA is negatively charged, so it will be attracted to the anode on the opposite side of the container. The green dyes are the mass standards and the blue dyes are the DNA samples.

Below is the Nanodrop One, the spectrophotometry device used at an absorbance of 260. This device allows scientists to asses the purity of the DNA.

The picture below is the results of the nano drop analysis. The data concluded that the sample had a 598 ng/uL mass, an A260/A280 range of 1.42, and an A260/230 range of 0.55. Although the sample isn’t completely pure, the data shows that the first hump is around the same range as a purified sample.

Below is the data for the agarose gel run. The bright lines are due to the Ethidium Bromide, and they are different widths due to the different mass standards used. For example, we can conclude that the DNA sample in Lane 5 is greater than 3.1 ng/uL when compared to the ’15’ standard. The lighter DNA smear in Lane 8 is due to the small amount of DNA used, due to complications. This procedure is explained in the conclusion.

Storage:

For storage, the micropipette tips were ejected and the pipettes were placed on the rack. The DNA samples were placed back into the ice cooler, and the agarose gels were placed back into the fridge.

Conclusions:

In conclusion, we had a successful gel run, and we found DNA! Although we had some tricky beginnings, we worked together in order to solve the problem. To start, our 9 uL DNA and 1 uL buffer mixture would not stay in Well 1. We resulted this failure to an air bubble, either in the pipette or the well, as every time we attempted to place sample in the well it would disperse into the buffer above. We solved this problem by using 2 uL of DNA, 2 uL of loading dye, and 6 uL of DI water. Although the sample appeared lighter on the gel scan, we found light markings of DNA and compared it to the results of the other group, Group 4. As for the nano drop analysis, we found our DNA sample to have a 598 ng/uL mass, A260/A280 range of 1.42, and an A260/230 range of 0.55. Although this reading wasn’t completely ideal, the first hump did come close to ideal values, as Mike explained. Overall, we learned informative skills that will come into play in advanced researched, and learned some of the why’s behind this process.

Future Steps:

In the future, I would have checked our wells in the agarose gel, as Lane 1 was problematic. Dr. Adair hypothesized that there was a bubble in the well, due to our issues with injecting the 10 uL DNA sample into the well. She advised that we pipette up and down in order to remove any bubbles.