4/20 Planning Gradient PCR

Today we planned our next steps of the experiment. We are going to preform a gradient PCR reactions to determine the true optimal temperature to get the Rubisco primers to cut our food samples. We decided that will will do a gradient PCR reaction with Cereal D because Cereal D was cut the best by the Rubisco Primers (see gel image below).

annotated gel of test foods 1

We made the following master mix for 10x reactions:

1x (uL) 10x (uL)
H2O 19 190
10x 2.5 25
DNTP .5 5
Forward Primer 1 10
Reverse Primer 1 10
Taq .125 1.25
*take out one reaction for neg control*
DNA 1 9
Total 25  250
We researched the listed optimal annealing temperature for our Rubisco primers, and found the temperature to be 41.7 degrees Celsius. Rubisco has the binding sites and length of the amplicon as shown in this image:
rubisco primer info
We are running a 43-60 degrees Celsius gradient. Our eight reactions will be placed, staring in column 3 and ending in column 10, in the PCR machine. Our samples will be annealed from 44.5-58.6 degrees Celsius.
gradient location
We then ran the PCR gradient following this procedure:
gradient pcr reaction protocol

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