5 26 2015 Oly cDNA/Primer Check

Today I ran a qPCR using the Actin, HSP70, and Glutamine synthetase primers used by the 310 class on 6 cDNA samples prepared by Sam last week. You can read about it here. I made fresh working stocks from the original primer stocks. As per Sam's suggestion I diluted the cDNA to a 1:4 dilution to save on cDNA so I can run multiple primer checks.

Primer Working Stocks:

Actin

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

HSP70

HSP70_gigas_F	GTTCCGATTTGTTCCGTGCC	KK		20			C.gigas	HSP70
HSP70_gigas_R	TTGTCGCCATTTTCCTCGCT	KK		20			C.gigas	HSP70

Glutamine synthetase

Gluta_F	ATTCCCTCCCCTGATCCGTCCACCCA						O.lurida	Glutamine synthetase
Gluta_R	TGGGTGGACGGATCAGGGGAGGGAAT						O.lurida	Glutamine synthetase

Working Stock Calculations:

	ul
Primer	10
Nuclease free H20	90

cDNA Dilution Calculations:

cDNA 1:4 Dilution
cDNA	10
Nuclease free H20	30
Final Volume	40

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:

	Volume	Reactions X12
Ssofast Evagreen MM	10	220
FWD Primer	0.5	11
REV Primer	0.5	11
Nuclease Free H2O	8	176
cDNA	1

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. 

Plate Layout:

Actin	Actin	HSP70	HSP70	Gluta	Gluta
7	8	9	10	11	12
C+	NTC	C+	NTC	C+	NTC
NT1	NT1	NT1	NT1	NT1	NT1
HT1	HT1	HT1	HT1	HT1	HT1
ST1	ST1	ST1	ST1	ST1	ST1
NC1	NC1	NC1	NC1	NC1	NC1
HC1	HC1	HC1	HC1	HC1	HC1
SC1	SC1	SC1	SC1	SC1	SC1
NTC	NTC	NTC	NTC	NTC	NTC

The results from the qPCR are below:

Actin Amp

Actin Melt Curve

HSP70 Amp

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.