Purpose/Objectives:

• Today in lab students were expected to complete the Polymerase chain reaction (PCR) protocol using the Cox1 forward and reverse primers, as well as preparing the agragose gel for the Gel Electrophoresis Protocol for next week’s lab.

Procedure:

PCR Procedure

• Determine the volumes of 2x Master Mix, DNA template, primers and water to add to our positive control, negative control and soil DNA tube for PCR as a class.
• Clean the lab table with bleach and wear gloves as it is important to carry out the preparation of the tubes for PCR under an aseptic environment.
  • Also, ethidium bromide is dangerous to skin contact, so be extra careful when using the compound, and wear gloves!
• Label the 3 tubes, each containing 12.5µl of 2x Master Mix, to differentiate the negative control tube, positive control tube, and the soil DNA tube.
  • In my group’s case: “Pos, Neg, DNA”
  • Our tubes were labeled: 7, SAM
• Use a p10 micropipette to transfer 5µl of DNA template to the positive control tube.
• Use a p10 micropipette to transfer 5µl of soil DNA to the soil DNA tube. Ensure that no DNA template is added to the negative control tube.
  • As stated in the tube’s name, this would ruin the entire procedure as this tube is meant to hold everything except DNA.
• Using a p10 micropipette, transfer 1µl of Cox1 primers into all three tubes.
• Micropipette 11.5µl of water into the negative control tube and 6.5µl of water each into the positive control tube and the soil DNA tube. Therefore, each of the 3 tubes should contain a total of 25µl of solution.

Gel Electrophoresis Protocol

• On a piece of weighing paper on an electronic balance, weigh out 0.6g of agarose gel powder.
• Transfer the weighed gel powder into an Erlenmeyer flask.
• Measure out 40ml of 1xTAE using a measuring cylinder.
• Add the measured 1xTAE to the Erlenmeyer flask containing agarose powder.
• (GENTLY) Cap the Erlenmeyer flask and weighing paper.
  • Capping the flask loosely is imperative, as this will prevent explosion when the flask is heated in the following step.
• Heat the flask in a microwave for 120s at power 7 so that the agarose gel powder dissolves completely in the solution.
  • Remove flask with hot pads to prevent burns.
• Place the flask in a water bath for 5 minutes at 55 degrees Celsius.
• In the meantime, assemble the gel plate and label the plate with the group number.
  • Room: A127, Name: SAM,Group 7, Dr: Adair 1106-22 in my group’s case.
• Once 5 minutes is over, remove the flask from the water bath.
• Add 2 ul ethidium bromide to the solution.
• Place the gel comb inside the tray.
• Carefully pour the gel into the assembled gel plate.
• Leave the gel to cool and solidify for 25-30 minutes.
  • You will know the gel is set properly once it begins to turn foggy from its previously transparent color.
• Give your gel plate to Will for safe keeping till the next lab.
  • ALWAYS CLEAN YOUR LAB AREA BEFORE LEAVING THE CLASS….
  • unless you WANT to lose points off your lab grade…

Data/Obeservations:

This table details the specific measurements my group used to complete the PCR protocol.

A photo of my group’s gel plate with our identification details.

Conclusion:

• This lab was very interesting. Aside from a good 5 minutes of Ahkan and my groupmate trying to explain to me the process of how to calculate the correct measurements, and despite the fact that only one of my groupmates was at the lab due to the other having a sickness, I feel proud that my groupmate and I were able to finish the lab and record all of our results.
• Our favorite part was making the agarose gel because that was the part that was most exciting to us while completing the prelab and we couldn’t wait to do it ourselves today.
• I am very eager to see if we will be able to amplify our DNA next class, although I am still concerned about the purity of our DNA sample, as well as the accuracy of its total content concentration.
• Other than that, I enjoyed this lab, and I feel as if my groupmate and I did not commit any errors while following both protocols.