Wednesday, May 27, 2015

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.

VolumeReactions X18
Ssofast Evagreen MM 10180
FWD Primer0.59
REV Primer0.59
Nuclease Free H2O8144
cDNA1

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:
ActinActinHSP70HSP70
1234
NTCNTCNTCNTC
NT1 NT1 NT1NT1
HT1HT1HT1HT1
ST1ST1ST1ST1
NC1NC1NC1NC1
HC1HC1HC1HC1
SC1SC1SC1SC1
NTCNTCNTCNTC
qPCR Program:
Sybr New Plate+Sybr cDNA 60 melt 2 Read
StepTemperatureTime
Initiation95 C10 min
Elongation95 C30 sec
55 C1 min
Read
72 C30 sec
Read
Repeat Elongation 39 times
Termination95 C1 min
55 C1 sec
Melt Curve Manual ramp 0.2C per sec Read 0.5 C55 - 95 C30 sec
21 C10 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.

1 comment: