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:

	Volume	Reactions X12
Ssofast Evagreen MM	10	280
FWD Primer	0.5	14
REV Primer	0.5	14
Nuclease Free H2O	8.5	238
RNA	0.5

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.

9	10	11	12
C-	C+	C-	C+
42715HM1	42715SM1	42715HT1	42715ST1
42715HM2	42715SM2	42715HT2	42715ST2
42715HM3	42715SM3	42715HT3	42715ST3
42715HM4	42715SM4	42715HT4	42715ST4
42715HM5	42715SM5	42715HT5	42715ST5

I ran the following program:

Sybr New Plate+Sybr cDNA 55 melt 2
Step	Temperature	Time
Initiation	95 C	10 min
Elongation	95 C	15 sec
	55 C	15 sec
Read
	72 C	15 sec
Read
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
End

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.