Because we had a lot of success with the Rubsico PCR gradient, we’ve decided to complete a PCR gradient with Dr. Todd’s GMO primers.
We made the following master mix for 10x reactions:
|1x (uL)||10x (uL)|
|*take out one reaction for neg control*|
The desired amplicon length is 125 base-pairs. We researched the optimal annealing temperature for the primers and found that to be 50.59 degrees Celsius. We are running a 46-60 degree Celsius gradient. Our eight reactions will be placed, staring in column 4 and ending in column 11, in the PCR machine. Our samples will be annealed from 48.3-59.7 degrees Celsius.
We then ran the PCR gradient following this procedure: