Purpose 
The purpose of this lab was to begin the DNA application stage of our protocol. We began this procedure by becoming familiar with Cox1 reverse and forward primary. Using these processes, amplificication could be completed: template DNA with nucleotides, buffer, DNA polymerase, an d primates. Once we had prepared our negative, positive, and sample tube, we created the agarose gel that we will be using next week for the amplified DNA.

Procedure 

◦ Negative control tube

‣ Add 1uL primer and 11.5 uL water

‣ Label tube “negative”

◦ Positive control

‣ Add 12.5 uL of 2x Master Mix

‣ Add 5uL of DNA template

‣ 1 uL primers ‣ Add 6.5 uL water

‣ Label tube “positive”

◦ Sample tube

‣ Add 12.5 uL 2x Master Mix

‣ Add 5uL DNA Template and 1 uL of primer

‣ Add 6.5 uL of water

‣ Label tube “sample”

◦ Obtain Erlynmeyer flask and place in freezer for 5 minutes at 4 degrees C

◦ Add ethidium bromide to solution

◦ Pour into assembled gel tray with comb

◦ Label tray with piece of labeled tape

◦ Allow gel to solidify for 30 minutes

◦ Record location of test tube on test rack

• Data and Observations 

◦  Positive Control stored in C1 labels with a “P1”

◦ Negative control stored in C2 labeled with “N2”

◦ Sample tube stored in C3 labeled “S1”

◦ COX-1 forward primary: 5′ ATGTGAGTTGATTTTATAGAGCAGA-3′

◦ COX-1  reverse primer: 5′- GGDATACCRTTCATTTT-3′

◦ Recipe for 40mL of 1.5% TAE ‣ (40)(0.015) = ).6 grams of agarose needed

◦ Gel tray labeled “21-1 TCA” and left to solidify

Conclusion 

Despite the fact that we have not yet seen the effects of the COX1 primers, our class is indeed confident that we will find results. Nevertheless, if we do not find results, we can use the positive and negative control groups to help us modify our protocol. We ran out of time to remove the comb from the gel because the gel had to solidify for 30 minutes. In the future, we will be running the electrophoresis gels. This will allow us to examine the DNA sequences that were amplified. The ethidium bromide solution will be essential because this is what allows the sequences to be examined. In the following lab, we will be using the prepared controls and sample into the solidified gel and continue our protocol.