First, we reviewed the gel results of the GMO primer gradient reactions, which was pretty successful. Even though the negative control was contaminated, there was good amplification in the higher temperature reactions. To fix the issue with the contaminated negative control, we will use new water in our future reactions. We’ve concluded that Cereal D was best amplified at 57.8 degrees Celsius. Earlier, we concluded that the Rubisco primers best annealed at 57.3 degrees Celsius, so we will set both PCR reactions to 58 degrees Celsius for our next step.
For our next step, we will have 12 total reactions: a 10 log ladder, two negative controls, the five test foods with Rubisco primers, and the five test foods with GMO primers. We will create a master mix for 14 reactions that contains: 210μL (new) water, 35μL 10x buffer, 7μL dNTPs, and 1.75μL taq. We will separate the master mix into two equal parts and add 7μL of the Rubsico primers into one tube of master mix and add 7μL of the GMO primers into the second tube of master mix. Then we will will pipet 1μL of each test food into separate PCR tubes twice; therefore, we will have two tubes containing 1μL of each test food.
The following reactions were made:
The 12 tubes were placed in a PCR machine that followed this process:
- 95 degrees for 2 mins
- 35 cycles of:
- 95 degrees for 1 min
- 58 degrees for 1 min
- 68 degrees for 2 min
- 68 degrees for 10 mins
- Hold at 4 degrees