Wednesday, August 19, 2015

8 18 2015 18s/28s primer check qPCR

While looking for new normalizing primers to add to the EF1d and actin that I already have I've developed primers for 18s and 28s. The 18s are based on sequences for Ostrea conchaphila. The 28s primer is developed from the 28s sequence from the O.lurida Transcriptome version 3. I tested these primers on the mechanical stress samples in a 1:9 dilution.

Primers:
170228s_3_FWDTAAGGCCAGTGTGGGAGAGAJH8/12/2015205559.88O.lurida"28S ribosomal protein S5, mitochondrial (MRP-S5) (S5mt)"Q5REJ1
170128s_3_REVCGCCTCACTTTTTGTGCCTCJH8/12/2015205560.04O.lurida"28S ribosomal protein S5, mitochondrial (MRP-S5) (S5mt)"Q5REJ1
170018s_2_FWDCTCGATTCCCTTTCCCCCACJH8/12/2015206060.11O.conchaphila18s Ribosomal RNAEF035117.1
169918s_2_REVGCCCGATCTCTTTCCACCTCJH8/12/2015206060.18O.conchaphila18s Ribosomal RNAEF035117.1
169818s_1_FWDTTCCTTTTCCCCCACTCAGCJH8/12/2015205559.89O.conchaphila18s Ribosomal RNAEF035118.1
169718s_1_REVCGCTTCACTCGCCGTTACTAJH8/12/2015205560.18O.conchaphila18s Ribosomal RNAEF035118.1

Reagent Table:
VolumeReactions X10
Ssofast Evagreen MM 10100
FWD Primer0.55
REV Primer0.55
PCR H2O550
1:9 cDNA4
  1. Added reagents from greatest to least volume
  2. Vortexed
  3. Centrifuged briefly
  4. Pipetted 16 ul Master Mix to each tube
  5. Pipetted 4 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
Table Layout:
18s_118s_228s_3
12345
DNased 42215 HM1 1DNased 42215 NM1 1DNased 42215 SM1 118s_1_NTC18s_1_NTC
DNased 42215 HM1 2DNased 42215 NM1 2DNased 42215 SM1 218s_2_NTC18s_2_NTC
DNased 42215 HM1 3DNased 42215 NM1 3DNased 42215 SM1 328s_3_NTC28s_3_NTC
DNased 42215 HM1 4DNased 42215 NM1 4DNased 42215 SM1 4
DNased 42215 HM1 5DNased 42215 NM1 5DNased 42215 SM1 5
DNased 42215 HM1 6DNased 42215 NM1 6DNased 42215 SM1 6
DNased 42215 HM1 7DNased 42215 NM1 7DNased 42215 SM1 7
DNased 42215 HM1 8DNased 42215 NM1 8DNased 42215 SM1 8
18s_1
All
NTC
18s_2
All
NTC
28s_3
All
NTC

The 18s primers each have issues. The first has seemingly random amplification and poor melt curves. The second seems to autoamplify and has NTCs with amplification equal to the samples. 
The 28s primer looks great. It has strong amplification with only primer dimers appearing in the NTCs. I think this should be used for the next normalizing primers. 
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8 18 2015 EF1d CTM2 reps qPCR

Today I ran replicates of all populations and all treatments to generate data about expression values. Here I ran EF1d. 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:
1682EF1d_FWDGAACTGCCCACTGATTTGCCJH7/22/2015205560O.luridaElongation factor 1-delta (EF-1-delta)A5D989
1681EF1d_REVTGTGGGGTGAAACACGTTGAJH7/22/2015205060O.luridaElongation factor 1-delta (EF-1-delta)A5D989

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
Amplification
Melt Curve
NTCs
Amplification
Melt Curve

The amplification and melt curves look great. There is no appreciable amplification in the NTCs.

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

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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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