8 10 2015 GRB2 Mech Stress 2 rep qPCR

Today I ran mechanical stress samples in replicates of two to generate data about expression values. Here I ran GRB2 . I ran NTCs on a separate plate using the same reaction volume and 9 ul of PCR water.

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 X58
Ssofast Evagreen MM	10	580
FWD Primer	0.5	29
REV Primer	0.5	29
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:

Rep 1	Rep 1	Rep 1	Rep 2	Rep 2	Rep 2
1	2	3	4	5	6
DNased 42215 HM1 1	DNased 42215 NM1 1	DNased 42215 SM1 1	DNased 42215 HM1 1	DNased 42215 NM1 1	DNased 42215 SM1 1
DNased 42215 HM1 2	DNased 42215 NM1 2	DNased 42215 SM1 2	DNased 42215 HM1 2	DNased 42215 NM1 2	DNased 42215 SM1 2
DNased 42215 HM1 3	DNased 42215 NM1 3	DNased 42215 SM1 3	DNased 42215 HM1 3	DNased 42215 NM1 3	DNased 42215 SM1 3
DNased 42215 HM1 4	DNased 42215 NM1 4	DNased 42215 SM1 4	DNased 42215 HM1 4	DNased 42215 NM1 4	DNased 42215 SM1 4
DNased 42215 HM1 5	DNased 42215 NM1 5	DNased 42215 SM1 5	DNased 42215 HM1 5	DNased 42215 NM1 5	DNased 42215 SM1 5
DNased 42215 HM1 6	DNased 42215 NM1 6	DNased 42215 SM1 6	DNased 42215 HM1 6	DNased 42215 NM1 6	DNased 42215 SM1 6
DNased 42215 HM1 7	DNased 42215 NM1 7	DNased 42215 SM1 7	DNased 42215 HM1 7	DNased 42215 NM1 7	DNased 42215 SM1 7
DNased 42215 HM1 8	DNased 42215 NM1 8	DNased 42215 SM1 8	DNased 42215 HM1 8	DNased 42215 NM1 8	DNased 42215 SM1 8

All

Amplification

Melt Curve

NTCs

Amplification

Melt Cuve

The amplification looks good and the melt curves look good. There's nothing significant in the NTCs. I added the Ct values to the rep comparison spreadsheet. You can see the absolute value difference between reps below. 

Rep Diff
1.87
2.26
1.2
1.05
1.56
0.84
0.94
1.25
1.19
0.19
1.85
0.75
0.28
1.27
0.07
0.93
1.67
0.28
1.08
1.33
6.34
1.45
0.91
0.1

There are 14 out of 24 samples that are more than 1 ct difference.  These samples would definitely need to be re run. 

You can see the raw data here and NTCs here.