DNA extraction was performed with our two mutant and two wild type plants. We followed the typical DNA extraction protocol which we previously performed. Once DNA was extracted, A typical PCR protocol was set up in the PCR thermocycler. This protocol called for the first step of 95 °C for 30 seconds to allow time for the PCR tubes to be placed into the thermocycler. Step 2 was the melting step which was set to 95 °C for 15 seconds. Step three was the annealing step, which we found by subtracting ~5 °C from the lower Tm of our two primers which was 59.4 °C, so this step was set to 54.4 °C for 30 seconds. The fourth step was the extension step, which we set to 72 °C for one minute. Step five called for a GoTo step to step to, to repeat steps 2-4 for 29 times. Finally, step six was the end step. These steps were input into the thermocycler and our program was saved as “MHN” in the class folder. After the program was set up, our DNA product from extraction was quantified using Nanodrop as done in previous labs.
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