5 27 2015 Actin HSP70 re run

After reviewing yesterdays qPCR run with Steven, it was decided that I should re run the Actin and HSP70 qPCR again but with a higher annealing temperature. I've been using 55 C for previous runs but was having some issues with products and melt curves.

Master Mix Reagent Table.

	Volume	Reactions X18
Ssofast Evagreen MM	10	180
FWD Primer	0.5	9
REV Primer	0.5	9
Nuclease Free H2O	8	144
cDNA	1

1. Added each from greatest volume to least to make the master mix for each primer. 

2. Pipetted 19 ul master mix into each well of a qPCR partial plate

3. Added 1 ul sample to each tube

Plate Layout:

Actin	Actin	HSP70	HSP70
1	2	3	4
NTC	NTC	NTC	NTC
NT1	NT1	NT1	NT1
HT1	HT1	HT1	HT1
ST1	ST1	ST1	ST1
NC1	NC1	NC1	NC1
HC1	HC1	HC1	HC1
SC1	SC1	SC1	SC1
NTC	NTC	NTC	NTC

qPCR Program:

Sybr New Plate+Sybr cDNA 60 melt 2 Read
Step	Temperature	Time
Initiation	95 C	10 min
Elongation	95 C	30 sec
	55 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

Results:
Actin Amplification

Actin Melt Curve

 HSP70 Amplification

 HSP70 Melt Curve

The No template controls were clean and the amplification clear with good melt curves. Overall this looks really good. My next step is to test the new primers that came in today. I'll post tomorrow about what they look like.

You can see the raw qPCR data file here.