4/21 Gradient Reactions for GMO Primers

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)
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

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.

gradient-of-gmo.jpg

We then ran the PCR gradient following this procedure:

gmo-gradient-pcr-procedure.jpg

 

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