4/26 Cereals A and B DNA Extraction and PCR Redo

As seen in the gel below, cereals A and B have degraded DNA; however, cereals C, D, and E had good amplification with Rubisco Primers. Unfortunately no primers or amplification appeared in the rows with test cereals and GMO primers. To fix these issues, we will do a new DNA extraction of cereals A and B. We will also redo the PCR reactions with cereals C, D, and E with both the Rubisco and GMO primers.

gel with both primers and optimal temps trial 1

Rubisco and GMO with Optimal Temperatures Gel 

We followed the same procedure as “Preparing the Negative Control” in the protocol to extract DNA from cereals A and B.

try 2 dna extraction from oats

DNA Extraction Procedure

The same master mix as all previous reactions was made, but this time we made it for 10 Rx. Then we created these 8 reactions below:

optimal temp pcr contents

Then we ran the PCR under the following conditions:

  • 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
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