8 14 2015 TLR CTM reps qPCR 2

Today I ran replicates of all populations and all treatments to generate data about expression values. Here I ran TLR2.1.

Primers:

1630	TLR2.1_FWD	ACAAAGATTCCACCCGGCAA	JH	5/21/2015	20	55		O.lurida	Toll-like receptor 2 type-1	Q9DD78
1629	TLR2.1_REV	ACACCAACGACAGGAAGTGG	JH	5/21/2015	20	55		O.lurida	Toll-like receptor 2 type-1	Q9DD78

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 

NTCs

The amplification curves look good. there's some minor amplification in the NTCs but its not appreciable compared to the samples. The melt curve for it falls in an odd place so I'm not sure what to make of it. 

You can see the raw ct values here and the raw data here.