Thursday, May 7, 2015

5 6 2015 qPCR Primer Dimer check

Yesterday I ran another qPCR on both sets of isolated RNA to determine whether the samples were truly contaminated with gDNA or if they were producing primer dimers. This required that we look at the melt curve to determine the size of the products being created.

The qPCR reagent table:
VolumeReactions X12
Ssofast Evagreen MM 10280
FWD Primer0.514
REV Primer0.514
Nuclease Free H2O8.5238

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.

qPCR protocol:

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

Table Layout.


I ran the following program:

Sybr New Plate+Sybr cDNA 55 melt 2
Initiation95 C10 min
Elongation95 C15 sec
55 C15 sec
72 C15 sec
Repeat Elongation 40 times
Termination95 C1 min
55 C1 sec
Melt Curve Manual ramp 0.2C per sec Read 0.5 C65 - 95 C30 sec
21 C10 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

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