Saturday, August 15, 2015

8 15 2015 Actin CTM2 reps qPCR

Today I ran replicates of all populations and all treatments to generate data about expression values. Here I ran Actin. To save on cDNA I stopped running full replicates of the Control and Temperature samples with the mech stress samples. I run partials made up of 8 samples for Control and 8 of Heat. 

Primers:
1505Ol_Act_FGACCAGCCAAATCCAGACGABC6/13/2012205560O.luridaActin, adductor muscle
1504Ol_Act_RCGGTCGTACCACTGGTATCG6/13/2012206060O.luridaActin, adductor muscle 
Reagent Table:

VolumeReactions X80
Ssofast Evagreen MM10800
FWD Primer0.540
REV Primer0.540
1:9 cDNA9
  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:
StepTemperatureTime
Initiation95 C10 min
Elongation95 C30 sec
60 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

Plate Layout:
123456789
DNased 42215 HM1 1DNased 42215 NM1 1DNased 42215 SM1 1DNased 42215 HM1 1DNased 42215 NM1 1DNased 42215 SM1 1DNased 42215 NC1DNased 42215 NT1 1NTC
DNased 42215 HM1 2DNased 42215 NM1 2DNased 42215 SM1 2DNased 42215 HM1 2DNased 42215 NM1 2DNased 42215 SM1 2DNased 42215 NC2DNased 42215 NT1 2NTC
DNased 42215 HM1 3DNased 42215 NM1 3DNased 42215 SM1 3DNased 42215 HM1 3DNased 42215 NM1 3DNased 42215 SM1 3DNased 42215 NC3DNased 42215 NT1 3NTC
DNased 42215 HM1 4DNased 42215 NM1 4DNased 42215 SM1 4DNased 42215 HM1 4DNased 42215 NM1 4DNased 42215 SM1 4DNased 42215 NC4DNased 42215 NT1 4NTC
DNased 42215 HM1 5DNased 42215 NM1 5DNased 42215 SM1 5DNased 42215 HM1 5DNased 42215 NM1 5DNased 42215 SM1 5DNased 42215 NC5DNased 42215 NT1 5
DNased 42215 HM1 6DNased 42215 NM1 6DNased 42215 SM1 6DNased 42215 HM1 6DNased 42215 NM1 6DNased 42215 SM1 6DNased 42215 NC6DNased 42215 NT1 6
DNased 42215 HM1 7DNased 42215 NM1 7DNased 42215 SM1 7DNased 42215 HM1 7DNased 42215 NM1 7DNased 42215 SM1 7DNased 42215 NC7DNased 42215 NT1 7
DNased 42215 HM1 8DNased 42215 NM1 8DNased 42215 SM1 8DNased 42215 HM1 8DNased 42215 NM1 8DNased 42215 SM1 8DNased 42215 NC8DNased 42215 NT1 8
All 
Amplification
Melt Curve
NTCs
Amplification
Melt Curve
The amplification curves look good but the melt curve has a double bump which looks terrible.  I'm not sure why this happened. It appears in all the samples. 

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

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