Gel Electrophoresis Lab 2/21/19
Rationale
The goal of today’s lab was to determine if there is any DNA in the samples and if so, then to find out how much DNA is in the sample. This was done by running the samples through the electrophoresis gel, then using the nanodrop and Gel Doc EZ System.
Experimental Design
Preparing the DNA
The gels made from last week had the combs removed from them so that 8 wells were open. The gel had a 1x (900ul of water and 100ul TAE buffer) TAE solution added to it so that the electric current could be carried and the pH kept constant. The DNA samples needed to be mixed with a loading buffer so that the density would increase and it would sink. 9uL of DNA sample was mixed with 1uL of loading buffer to create a 10% solution. This was spun by using a miniature centrifuge. The solution was then placed into the wells of the gel by using a 10uL micropipette. The first well was a test run in order to determine if the gel comb being placed backward would produce a faulty gel. The second and third gels were filled with the 31ng/uL and 250ng/uL mass standards. The last wells were filled with the DNA samples.
Running the DNA
After the wells were filled, then the gel was placed into a gel electrophoresis box. The wells were opposite of the positive electrodes because DNA is negatively charged. The electrophoresis box was turned on at 100 volts for 20 minutes set on the constant mode. After the time was over then the gels were taken to the third floor in order to use the nanodrop and Gel Doc EZ system.
Nanodrop
The nanodrop was calibrated by placing water in the pedestals and running it normally. The water was wiped off and the samples were placed into the pedestals individually. Each sample had roughly a 1uL sample. A good ratio of pure DNA to protein is 1.80. The nanodrop is used to determine the purity of the DNA using light spectrophotometry.
Gel Doc EZ System
After the nanodrop, the gels were put in the Gel Doc EZ System in order to illuminate the ethidium bromide in the DNA sample under ultraviolet light. The test used the ultraviolet tray. The test results were saved and emailed to the students who had samples in the gel. This test is also used to determine the purity of a sample by the brightness of the band under ultraviolet light.
Observation and Analysis
Preparing the DNA
When I was transferring the 10uL sample into the well, I had accidentally come in at an angle and I either punctured the well or I missed the well. Either way the results were mostly unaffected but I had less loading buffer than the rest of the samples in the well.
The gel electrophoresis box. From left to right, the wells are 1-8.
Gel with DNA samples after the 20 minute period of 100 volts
Running the DNA
A table of the well designation
| Well 1 | Well 2 | Well 3 | Well 4 | Well 5 | Well 6 | Well 7 | Well 8 |
|
test run to determine the well |
31ng/uL standard mass | 250ng/uL standard mass | Quinn’s Sample | Britney’s Sample | Chris’s Sample | Garret’s Sample | Caleb’s Sample |
Nanodrop
The number from the nanodrop machine-
| ng/uL | 250.2 |
| A260/A280 | 1.47 |
| A260/A230 | 0.68 |
These numbers show that the DNA is fairly pure but it could be better.
Gel Doc EZ System
The wells 4-7 are chelex procedure samples. Well 8 is the soil DNA sample purification method. There is DNA in each well, however the red bands in well 3 (standard mass 250ng/uL) and 8 signify that the sample is saturated or it is above 250bp.
Conclusion
In this lab, our DNA samples were determined to have DNA in them and a rough estimation of how much DNA. This will enable us to compare the different samples collected from each student.
Next Step
For the next step in the lab, we will continue to observe our DNA samples and determine how much DNA we have in. We will continue to move towards writing a protocol article for extracting DNA in the soil of the trees on Baylor’s campus so the ciliate biodiversity can be determined. We will do this by adding to our methods section.