7 28 2015 Normalizing Gene Primer Tests

In an effort to find a better normalizing primer than the Actin one I have, I developed 4 possible normalizing primers. Today I tested all 4 of them on a 1:9 dilution of cDNA. 

Primers:

1688	GAPDH_FWD	TGCTCCTTGCATTTCCGTCA	JH	7/22/2015	20	50	60	O.lurida	Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (EC 1.2.1.12)	Q6BMK0
1687	GAPDH_REV	CGCTCCTTCCAAGTCTCCAG	JH	7/22/2015	20	60	60	O.lurida	Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (EC 1.2.1.12)	Q6BMK0
1686	Act_FWD	GTTCTCCTGTCGGGTGGAAG	JH	7/22/2015	20	60	60	O.lurida	Actin	P53498
1685	Act_REV	CACCATGAAACGCCGACTTG	JH	7/22/2015	20	55	60	O.lurida	Actin	P53498
1684	EFG_FWD	ACCCAAGGACAGGGAGAGTT	JH	7/22/2015	20	55	60	O.lurida	"Elongation factor G, mitochondrial (EF-Gmt) (Elongation factor G 1, mitochondrial) (mEF-G 1) (Elongation factor G1) (Protein iconoclast)"	B3MK91
1683	EFG_REV	TGTGTCCCCTTGTTGCTCTC	JH	7/22/2015	20	55	60	O.lurida	"Elongation factor G, mitochondrial (EF-Gmt) (Elongation factor G 1, mitochondrial) (mEF-G 1) (Elongation factor G1) (Protein iconoclast)"	B3MK91
1682	EF1d_FWD	GAACTGCCCACTGATTTGCC	JH	7/22/2015	20	55	60	O.lurida	Elongation factor 1-delta (EF-1-delta)	A5D989
1681	EF1d_REV	TGTGGGGTGAAACACGTTGA	JH	7/22/2015	20	50	60	O.lurida	Elongation factor 1-delta (EF-1-delta)	A5D989

Reagent Table:

	Volume	Reactions X116
Ssofast Evagreen MM	10	1160
FWD Primer	0.5	58
REV Primer	0.5	58
1:9 cDNA	9

1. Added reagents from greatest to least volume
2. Vortexed
3. Centrifuged briefly
4. Pipetted 11 ul Master Mix to each tube
5. Pipetted 9 ul of 1:9 cDNA each column using a channel pipetter
6. Centrifuged plate at 2000 rpm for 1 minute
7. Ran Program Below

Program:

Step	Temperature	Time
Initiation	95 C	10 min
Elongation	95 C	30 sec
	60 C	1 min
Read
	72 C	30 sec
Read
Repeat Elongation 39 times
Termination	95 C	1 min
	55 C	1 sec
Melt Curve Manual ramp 0.2C per sec Read 0.5 C	55 - 95 C	30 sec
	21 C	10 min
End

Plate Layout:

EFG	EF1D	GAPDH	Actin
1	2	3	4
NTC	NTC	NTC	NTC
DNased 42215 HC8	DNased 42215 HC7	DNased 42215 HC6	DNased 42215 HC5
DNased 42215 NC8	DNased 42215 NC7	DNased 42215 NC6	DNased 42215 NC5
DNased 42215 SC8	DNased 42215 SC7	DNased 42215 SC6	DNased 42215 SC5
DNased 42215 HT1 8	DNased 42215 HT1 7	DNased 42215 HT1 6	DNased 42215 HT1 5
DNased 42215 NT1 8	DNased 42215 NT1 7	DNased 42215 NT1 6	DNased 42215 NT1 5
DNased 42215 ST1 8	DNased 42215 ST1 7	DNased 42215 ST1 6	DNased 42215 ST1 5
NTC	NTC	NTC	NTC

Results:

EFG

Amp

 Melt Curve

EF1d

Amp

 Melt Curve

GAPDH

Amp

 Melt Curve

Actin

Amp

 Melt Curve

The best primer looks like it EF1d as the curves are strong and the melt curve is tight. EFG is second best, good curves and ok melt curve. Actin is third best, amplification curves are weak as well as the melt curve. GAPDH failed to amplify. 

So I'm going to run EF1d tomorrow. 

You can see the raw data here.