Carter Pope
Professor John Paul
Molecular Ecology
11/10/18
Lab 11 Entry
Lupinus arboreus DNA PCR
On November 7th, I arrived at the lab to shift my focus towards running a PCR with ISSR primers for some Lupinus arboreus DNA samples that were from a previous class. I started by making three dilutions for each of my five Lupinus arboreus that I was given (making a total of 15 dilutions). In order to do this, I acquired fifteen 1.5 millimeter tubes and used a sharpie to label each tube with its correct sample ID and ‘1:10’ (each of the five samples had its unique ID). I then used a micropipette to transfer 45 microliters of water into each of the 15 labeled tubes. Next, I used a micropipette to transfer 5 microliters of the correct DNA sample into each of its three corresponding 1.5 millimeter tubes (making sure to change tips each time). Once my group completed these steps, we then put our dilutions into the correct tray for our professor to disperse evenly for each of the three different lab groups.
After our professor gave the lab groups the trays of every diluted sample, my lab table peers and I chose 3 different sets of the five DNA samples at random to work with a total of fifteen Lupinus arboreus DNA samples (For example, I chose CRA01-CRA05, PRK01-PRK05, and PSF01-PSF05) and recorded their unique ID and the date down on a piece of paper. I then got two strips of eight 0.2 millimeter tubes and labeled each tube lid with a number (starting with ‘1’ for the first tube and increasing in value with each tube until reaching the last tube and labeling it ’16’). I then labeled the side of each tube with the unique ID and put both my initials (CP) and our primer name (17898) on the side of the strip of tubes. I used a micropipette and filtered tips (making sure to change them after each transfer) to then transfer 1 microliter of each of 15 different diluted DNA samples into each of the correct corresponding 0.2 millimeter tubes that I just labeled in the previous step. Once I finished transferring the 1 microliter into the tubes, I put my tube strip on ice until my lab group finished.
During this time, a person in my lab group (Kayla V.) made a master mix for 80 reactions (60 for our group’s total number of samples plus an additional 20 just in case). I then used a micropipette to transfer 19 microliters of the master mix into each 0.2 millimeter tube in the strips (being careful to change the tips each time) and carefully mixed the solution by pipetting up and down slowly in the tube. The tube stips were then given to our professor, who would then put them into the PCR machine.