8 14 2015 GRB2 CTM reps qPCR

Today I ran replicates of all populations and all treatments to generate data about expression values. Here I ran GRB2.

Primers:

1612	GRB2_FWD	AACTTTGTCCACCCAGACGG	JH	5/21/2015	20	55		O.lurida	Growth factor receptor-bound protein 2 (Adapter protein GRB2) (Protein Ash) (SH2/SH3 adapter GRB2)	P62994
1611	GRB2_REV	CCAGTTGCAGTCCACTTCCT	JH	5/21/2015	20	55		O.lurida	Growth factor receptor-bound protein 2 (Adapter protein GRB2) (Protein Ash) (SH2/SH3 adapter GRB2)	P62994

Reagent Table:

	Volume	Reactions X80
Ssofast Evagreen MM	10	800
FWD Primer	0.5	40
REV Primer	0.5	40
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 using a  multichannel pipetter
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:

1	2	3	4	5	6	7	8	9	10
DNased 42215 HC1	DNased 42215 NC1	DNased 42215 SC1	DNased 42215 HT1 1	DNased 42215 NT1 1	DNased 42215 ST1 1	DNased 42215 HM1 1	DNased 42215 NM1 1	DNased 42215 SM1 1	NTC
DNased 42215 HC2	DNased 42215 NC2	DNased 42215 SC2	DNased 42215 HT1 2	DNased 42215 NT1 2	DNased 42215 ST1 2	DNased 42215 HM1 2	DNased 42215 NM1 2	DNased 42215 SM1 2	NTC
DNased 42215 HC3	DNased 42215 NC3	DNased 42215 SC3	DNased 42215 HT1 3	DNased 42215 NT1 3	DNased 42215 ST1 3	DNased 42215 HM1 3	DNased 42215 NM1 3	DNased 42215 SM1 3	NTC
DNased 42215 HC4	DNased 42215 NC4	DNased 42215 SC4	DNased 42215 HT1 4	DNased 42215 NT1 4	DNased 42215 ST1 4	DNased 42215 HM1 4	DNased 42215 NM1 4	DNased 42215 SM1 4	NTC
DNased 42215 HC5	DNased 42215 NC5	DNased 42215 SC5	DNased 42215 HT1 5	DNased 42215 NT1 5	DNased 42215 ST1 5	DNased 42215 HM1 5	DNased 42215 NM1 5	DNased 42215 SM1 5
DNased 42215 HC6	DNased 42215 NC6	DNased 42215 SC6	DNased 42215 HT1 6	DNased 42215 NT1 6	DNased 42215 ST1 6	DNased 42215 HM1 6	DNased 42215 NM1 6	DNased 42215 SM1 6
DNased 42215 HC7	DNased 42215 NC7	DNased 42215 SC7	DNased 42215 HT1 7	DNased 42215 NT1 7	DNased 42215 ST1 7	DNased 42215 HM1 7	DNased 42215 NM1 7	DNased 42215 SM1 7
DNased 42215 HC8	DNased 42215 NC8	DNased 42215 SC8	DNased 42215 HT1 8	DNased 42215 NT1 8	DNased 42215 ST1 8	DNased 42215 HM1 8	DNased 42215 NM1 8	DNased 42215 SM1 8

All 

Amplification

Melt Curve

NTCs

Amplification

Melt Curve

The amplification curves look good but the melt curve is troubling. I've never seen GRB2 have this double hump on its samples. The NTCS don't show any appreciable amplification. 

You can see the raw Ct values here and the raw data file here.