Lab Entry 5 – Gel Electrophoresis
A template organizing where the samples were placed was provided in which each of the samples for the table was designated a specific lane to be place in. The gels being used have two rows with 8 lanes in each. The top row of gel was occupied by other table-mates and a ladder while the bottom row consisted of my three samples and another ladder. The gel was loaded in accordance to this template.
With a 1% agarose gel with GelRed already cast, the gel tray was placed into the gel box with the top of the gel at the negative (black) end of the electrical connector. 1x TAE buffer was poured into the gel box so that there was enough to cover the gel by a couple millimeters.
On a piece of parafilm, 2.0 μl Loading Dye was dropped for the number of samples being used. In our case, we placed 12 drops on the parafilm to be used for each of the samples for the table. Approximately 3.0 μl of gDNA was added to the Loading Dye for each of the samples. This was done until all dots were completed with Loading Dye and gDNA.
The pipette was readjusted to 5.0 μl and then each drop was pipetted into the designated well on the gel for all of the samples of gDNA. Once all of the wells were filled with their designated samples, the lid was placed onto the gel box with positive and negative charges aligned and then turned on. The volts were set to 145 and the gel ran for approximately 16 minutes before the power was turned off.
The gel was then imaged using the Gel Doc EZ Imager.
For the gel used for the PCR electrophoresis, the gel previously used for the gDNA run was melted down in a microwave to be reused for the PCR run. The gel was placed into a beaker and placed in a microwave for approximately 25 seconds, allowing the solution to bubble for about 10 seconds. To the liquid, another 1 μl of GelRed was added to the beaker in case of any loss. The gel was then poured into the casting rig set up to make the gel with the two rows of wells using combs. The gel was left to harden for about 10 minutes to be used for the PCR electrophoresis run.
For the PCR electrophoresis run, the same procedure was performed as for the gDNA electrophoresis run. All of the measurements were the same as well as the entirety of the process of dying and placement of samples on the gel. The only difference was the addition of a negative control that was made in the previous post which was then placed into one of the wells on the gel. Voltage and time for the electrophoresis run were the same as for the gDNA run.
For the clean up process, we used RA02, RA03, JPCC, JPCD, JW01, JW02, & JW03. These lanes had the best results for banding based on the computer imaging of the PCR gel plate. A master mix of ExoSap solution was created for all of the table-mates. Enough solution was created so that 9 clean-ups could be performed from the best samples and extra solution in case it was needed. The master mix consisted of 95.31 μl water, 11 μl 10x buffer (Sap 10x), 3.96 μl SAP, and 1.98 μl Exo. 7.5 μl of PCR product was placed into 0.2 μl PCR tubes for each of the samples in addition to 12 μl of the ExoSap master mix. Once all tubes contained the necessary amounts of products, they were placed into a thermocycler to undergo the EXOSAP program which takes approximately 45 minutes.