Labwork (PCR)

Following DNA extractions, it was time for amplification of DNA through Polymerase Chain Reaction (PCR). PCR amplifies the DNA by denaturing, copying, and synthesizing over and over again. Denaturing occurs via temperature, first during the DNA extraction process, and then again using the Thermocline machine (or PCR machine). The thermocline helps to regulate temperature allowing for PCR to go through its different steps, such as synthesis or denaturing. Synthesis occurs using a special polymerase Taq.

Kabat et. al is a paper that developed 8 primers specifically for this species of milkweed, Asclepias syriaca. For my experiment I will be using 7 of these 8. These primers help to amplify microsatellites in this case. Microsatellites are repeated sections of DNA unique to each plant. Since my experiment is looking at clonality, microsatellite primers are a great way to determine if plants are clonal.  When analysis occurs, if plants are clonal the sections amplified by the primers will be identical. If they are not identical, they are not clones.

 

Though the PCR protocol was adapted by me during spring semester, new primers and a host of other problems made this step tricky. The first batch sent off for analysis came back blank, and the second had wonky results. A delay in shipment meant that only 3 of the 7 primers were available. In the last remaining weeks of July PCR was done on all 400 of the plants for 3 of the primers. All PCR products were shipped off for fragment analysis. Training began on using a fragment analysis software, so once results do come back analysis will be smoother. This was a slightly frustrating time of the experiment as not as much could be accomplished as was hoped, but sometimes that’s how scientific research goes.

Speak Your Mind

*