The qPCR reagent table:
|Ssofast Evagreen MM||10||280|
|Nuclease Free H2O||8.5||238|
For the qPCR I used Actin primers and a positive control from Fidalgo seed oysters extracted on 3/23/2015 with a concentration of 167.3 ng/ul. My no template control contained no DNA as to show there was no contamination in the Master Mix.
1. Added each from greatest volume to least to make the master mix.
2. Pipetted 19.5 ul master mix into each well of a qPCR partial plate
3. Added 0.5 ul sample to each tube
I ran the following program:
|Sybr New Plate+Sybr cDNA 55 melt 2|
|Initiation||95 C||10 min|
|Elongation||95 C||15 sec|
|55 C||15 sec|
|72 C||15 sec|
|Repeat Elongation 40 times|
|Termination||95 C||1 min|
|55 C||1 sec|
|Melt Curve Manual ramp 0.2C per sec Read 0.5 C||65 - 95 C||30 sec|
|21 C||10 min|
You can see the amplification curves below:
You can see the Melt curve below:
It appears that there is definite gDNA contamination in the samples which is a shame. To remedy this I will be running another round of DNase treatment on them today. I'll check them with another qPCR tonight. You can view the raw qPCR file here.