Tuesday, August 18, 2015

8 18 2015 CARM CTM2 reps qPCR

Today I ran replicates of all populations and all treatments to generate data about expression values. Here I ran CARM. 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:
1642CARM1_FWDTGGTTATCAACAGCCCCGACJH5/21/20152055O.luridaHistone-arginine methyltransferase CARM1 (EC 2.1.1.-) (EC 2.1.1.125) (Coactivator-associated arginine methyltransferase 1) (Protein arginine N-methyltransferase 4)Q6DC04
1641CARM1_REVGTTGTTGACCCCAGGAGGAGJH5/21/20152055O.luridaHistone-arginine methyltransferase CARM1 (EC 2.1.1.-) (EC 2.1.1.125) (Coactivator-associated arginine methyltransferase 1) (Protein arginine N-methyltransferase 4)Q6DC04

 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 SC1DNased 42215 ST1 1NTC
DNased 42215 HM1 2DNased 42215 NM1 2DNased 42215 SM1 2DNased 42215 HM1 2DNased 42215 NM1 2DNased 42215 SM1 2DNased 42215 SC2DNased 42215 ST1 2NTC
DNased 42215 HM1 3DNased 42215 NM1 3DNased 42215 SM1 3DNased 42215 HM1 3DNased 42215 NM1 3DNased 42215 SM1 3DNased 42215 SC3DNased 42215 ST1 3NTC
DNased 42215 HM1 4DNased 42215 NM1 4DNased 42215 SM1 4DNased 42215 HM1 4DNased 42215 NM1 4DNased 42215 SM1 4DNased 42215 SC4DNased 42215 ST1 4NTC
DNased 42215 HM1 5DNased 42215 NM1 5DNased 42215 SM1 5DNased 42215 HM1 5DNased 42215 NM1 5DNased 42215 SM1 5DNased 42215 SC5DNased 42215 ST1 5
DNased 42215 HM1 6DNased 42215 NM1 6DNased 42215 SM1 6DNased 42215 HM1 6DNased 42215 NM1 6DNased 42215 SM1 6DNased 42215 SC6DNased 42215 ST1 6
DNased 42215 HM1 7DNased 42215 NM1 7DNased 42215 SM1 7DNased 42215 HM1 7DNased 42215 NM1 7DNased 42215 SM1 7DNased 42215 SC7DNased 42215 ST1 7
DNased 42215 HM1 8DNased 42215 NM1 8DNased 42215 SM1 8DNased 42215 HM1 8DNased 42215 NM1 8DNased 42215 SM1 8DNased 42215 SC8DNased 42215 ST1 8

All
Amplification
Melt Curves
NTCs
Amplification
Melt Curves

The amplification and melt curves look great, definitely better than the failure on saturday. 

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