Primer Working Stocks:
|Ol_Act_F||GACCAGCCAAATCCAGACGA||BC||6/13/2012||20||55||60||O.lurida||Actin, adductor muscle|
|Ol_Act_R||CGGTCGTACCACTGGTATCG||6/13/2012||20||60||60||O.lurida||Actin, adductor muscle|
Working Stock Calculations:
|Nuclease free H20||90|
cDNA Dilution Calculations:
|cDNA 1:4 Dilution|
|Nuclease free H20||30|
I then made up enough master mix to run 22 reactions. I miscalculated the number of no template controls (NTCs) I needed. I made too much master mix for this run and will cut back on the next run.
Master Mix Reagent Table:
|Ssofast Evagreen MM||10||220|
|Nuclease Free H2O||8||176|
To make each master mix, 3 total with one mix for each set of primers, I added the volumes from greatest to least of the reagents.
Then I pipetted the master mixes into the plate followed by 1 ul of either water, positive control, or sample. I made duplicates of each sample to ensure the results. The positive control was from the NF31 Seed oyster DNA Isolation from 3/23/2015 with a concentration of 117.53 ng/ul.
The results from the qPCR are below:
Actin Melt Curve
HSP70 Melt Curve
Glutamine Synthetase Amp
Glutamine Synthetase Melt Curve
Something was wrong with the Positive control which failed to amplify with two of the three primers. It also appears that glutamine synthetase indescriminately amplifies as it amplified in all three of the NTCs. Finally it appears that only 1 of the HSP samples amplified early than the controls.
I can rerun this qPCR tomorrow with a better positive control but I believe the Actin and HSP70 primers still work well while the glutamine synthetase primer does not. I will begin running the new primers when they come in.
You can see the raw qPCR data here.