Saturday, August 15, 2015

8 15 2015 PGRP CTM2 reps qPCR

Today I ran replicates of all populations and all treatments to generate data about expression values. Here I ran PGRP. 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:
1632PGRP-S_FWDGAGACTTCACCTCGCACCAAJH5/21/20152055O.luridaPeptidoglycan recognition protein 1 (Peptidoglycan recognition protein short) (PGRP-S)O75594
1631PGRP-S_REVAACTGGTTTGCCCGACATCAJH5/21/20152055O.luridaPeptidoglycan recognition protein 1 (Peptidoglycan recognition protein short) (PGRP-S)O75594










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 HC1DNased 42215 HT1 1NTC
DNased 42215 HM1 2DNased 42215 NM1 2DNased 42215 SM1 2DNased 42215 HM1 2DNased 42215 NM1 2DNased 42215 SM1 2DNased 42215 HC2DNased 42215 HT1 2NTC
DNased 42215 HM1 3DNased 42215 NM1 3DNased 42215 SM1 3DNased 42215 HM1 3DNased 42215 NM1 3DNased 42215 SM1 3DNased 42215 HC3DNased 42215 HT1 3NTC
DNased 42215 HM1 4DNased 42215 NM1 4DNased 42215 SM1 4DNased 42215 HM1 4DNased 42215 NM1 4DNased 42215 SM1 4DNased 42215 HC4DNased 42215 HT1 4NTC
DNased 42215 HM1 5DNased 42215 NM1 5DNased 42215 SM1 5DNased 42215 HM1 5DNased 42215 NM1 5DNased 42215 SM1 5DNased 42215 HC5DNased 42215 HT1 5
DNased 42215 HM1 6DNased 42215 NM1 6DNased 42215 SM1 6DNased 42215 HM1 6DNased 42215 NM1 6DNased 42215 SM1 6DNased 42215 HC6DNased 42215 HT1 6
DNased 42215 HM1 7DNased 42215 NM1 7DNased 42215 SM1 7DNased 42215 HM1 7DNased 42215 NM1 7DNased 42215 SM1 7DNased 42215 HC7DNased 42215 HT1 7
DNased 42215 HM1 8DNased 42215 NM1 8DNased 42215 SM1 8DNased 42215 HM1 8DNased 42215 NM1 8DNased 42215 SM1 8DNased 42215 HC8DNased 42215 HT1 8








All
NTCs
The amplification and melt curves look good. There's no appreciable amplification in the NTCs and this bump is just noise. 

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