8 26 2015 28s CTM qPCR 1

Today I ran two reps of the 28s target, one on the opticon and one at the CFX. We may use this as a normalizing gene if the reads look consistent. This is the run on the opticon. 

Primers:

1702	28s_3_FWD	TAAGGCCAGTGTGGGAGAGA	JH	8/12/2015	20	55	59.88	O.lurida	"28S ribosomal protein S5, mitochondrial (MRP-S5) (S5mt)"	Q5REJ1
1701	28s_3_REV	CGCCTCACTTTTTGTGCCTC	JH	8/12/2015	20	55	60.04	O.lurida	"28S ribosomal protein S5, mitochondrial (MRP-S5) (S5mt)"	Q5REJ1

Reagent Table:

	Volume	Reactions X80
Ssofast Evagreen MM	10	800
FWD Primer	0.5	40
REV Primer	0.5	40
PCR H2O	5	400
1:9 cDNA	4

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 and melt curves look good. There's some minor amplification in the NTCs but it appears to be a random product larger than the target so there's no worry about it. 

You can find the raw ct value data here and the raw data here.