Following DNA extraction we will set-up our PCRs. Remember that we will be using PCR to determine if the CaMV 35S sequence is present that would indicate that the item has been genetically modified. Each of the four groups will set-up six (6) reactions. Use the following number scheme to label your tubes. Also be sure to include your initials, the date, and the lab section on the tubes. Make sure you use different pipette tips between samples! Also make sure that you do not disturb the InstaGene beads when transferring your DNA. When instructed, place your samples in the thermal cycler and execute the program “GMO.”
|Tube||Primer Mix (forward, reverse, loading dye)||DNA|
|1||22 ul Plant Primer Mix||3 ul non-GMO control DNA|
|2||22 ul GMO Primer Mix||3 ul non-GMO control DNA|
|3||22 ul Plant Primer Mix||3 ul test food DNA|
|4||22 ul GMO Primer Mix||3 ul test food DNA|
|5||22 ul Plant Primer Mix||3 ul GMO positive control DNA|
|6||22 ul GMO Primer Mix||3 ul GMO positive control DNA|
You will notice that each DNA sample is being amplified twice using two different master mixes, one plant and one GMO. The plant master mix contains primers that will amplify ~200 bp of a gene within the chloroplast DNA (cpDNA). Why do we include primers for this gene? This helps us to guard against false negative results. For example, we would be fairly confident that a food item did not contain GMO sequences if no bands were detected using the GMO primers, but the cpDNA band was detected. Conversely, if no bands were detected with either master mix we cannot be entirely confident in our results that the item is non-GMO. The lack of bands with both master mixes might be due to a poor DNA extraction.
Looking at the table, can you provide a reason why we are including non-GMO control DNA in the experiment? How does this make the experiment more reliable?
You will also notice that our DNA samples contain a GMO positive control. Discuss why this is useful to our experiment. If the GMO positive control yielded no bands with the GMO master mix, what might you conclude?
In general, why is it useful to include multiple controls when performing molecular genetic experiments? How might this influence the diagnosis of various human diseases?
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