Primer:

1606 | H2A_FWD | GCTGGGGTTTTTCTGGGTCT | JH | 5/21/2015 | 20 | 55 | O.lurida | Histone H2A | P02270 | |

1605 | H2A_REV | GGAACTACGCCGAGAGAGTG | JH | 5/21/2015 | 20 | 55 | O.lurida | Histone H2A | P02270 |

Reagent Table:

Volume | Reactions X116 | |

Ssofast Evagreen MM | 10 | 1160 |

FWD Primer | 0.5 | 58 |

REV Primer | 0.5 | 58 |

1:9 cDNA | 9 |

- Added reagents from greatest to least volume
- Vortexed
- Centrifuged briefly
- Pipetted 11 ul Master Mix to each tube
- Pipetted 9 ul of 1:9 cDNA each column using a channel pipetter
- Centrifuged plate at 2000 rpm for 1 minute
- Ran Program Below

Program:

Step | Temperature | Time |

Initiation | 95 C | 10 min |

Elongation | 95 C | 30 sec |

60 C | 1 min | |

Read | ||

72 C | 30 sec | |

Read | ||

Repeat Elongation 39 times | ||

Termination | 95 C | 1 min |

55 C | 1 sec | |

Melt Curve Manual ramp 0.2C per sec Read 0.5 C | 55 - 95 C | 30 sec |

21 C | 10 min | |

End |

Plate Layout:

1 | 2 | 3 | 4 | 5 | 6 | 7 |

DNased 42215 HC1 | DNased 42215 NC1 | DNased 42215 SC1 | DNased 42215 HT1 1 | DNased 42215 NT1 1 | DNased 42215 ST1 1 | NTC |

DNased 42215 HC2 | DNased 42215 NC2 | DNased 42215 SC2 | DNased 42215 HT1 2 | DNased 42215 NT1 2 | DNased 42215 ST1 2 | NTC |

DNased 42215 HC3 | DNased 42215 NC3 | DNased 42215 SC3 | DNased 42215 HT1 3 | DNased 42215 NT1 3 | DNased 42215 ST1 3 | NTC |

DNased 42215 HC4 | DNased 42215 NC4 | DNased 42215 SC4 | DNased 42215 HT1 4 | DNased 42215 NT1 4 | DNased 42215 ST1 4 | NTC |

DNased 42215 HC5 | DNased 42215 NC5 | DNased 42215 SC5 | DNased 42215 HT1 5 | DNased 42215 NT1 5 | DNased 42215 ST1 5 | |

DNased 42215 HC6 | DNased 42215 NC6 | DNased 42215 SC6 | DNased 42215 HT1 6 | DNased 42215 NT1 6 | DNased 42215 ST1 6 | |

DNased 42215 HC7 | DNased 42215 NC7 | DNased 42215 SC7 | DNased 42215 HT1 7 | DNased 42215 NT1 7 | DNased 42215 ST1 7 | |

DNased 42215 HC8 | DNased 42215 NC8 | DNased 42215 SC8 | DNased 42215 HT1 8 | DNased 42215 NT1 8 | DNased 42215 ST1 8 |

Results:

All samples

NTCs

These runs look good as well but with a dimer appearing in 1 of the 4 NTCs. This is ok because it is much smaller than the target gene and thus not a sign of contamination. To determine if this was useful for normalizing I ran an ANOVA and Tukey's HSD to determine significant differences between treatment and controls.

Call:

aov(formula = expression ~ Pop + Treat + Pop:Treat, data = rep2res2)

Terms:

Pop Treat Pop:Treat Residuals

Sum of Squares 5.114600e-24 4.006829e-22 1.928451e-22 2.578036e-21

Deg. of Freedom 2 1 2 35

Residual standard error: 8.582435e-12

Estimated effects may be unbalanced

> TukeyHSD(fit)

Tukey multiple comparisons of means

95% family-wise confidence level

Fit: aov(formula = expression ~ Pop + Treat + Pop:Treat, data = rep2res2)

$Pop

diff lwr upr p adj

N-H 0.9770758

S-H 0.9980605

S-N 0.9652703

$Treat

diff lwr upr p adj

**T-C 0.0258271**

$`Pop:Treat`

diff lwr upr p adj

N:C-H:C 0.9284828

S:C-H:C 0.9913900

H:T-H:C 0.1254093

N:T-H:C 0.8827253

S:T-H:C 0.4351436

S:C-N:C 0.9989003

H:T-N:C 0.7722933

N:T-N:C 0.9999997

S:T-N:C 0.9823553

H:T-S:C 0.4682695

N:T-S:C 0.9968805

S:T-S:C 0.8575230

N:T-H:T 0.7753255

S:T-H:T 0.9778512

S:T-N:T 0.9865781

This would not make a good normalizing gene as there is a significant difference between the treatment and control. There doesn't appear to be any significant differences between populations or between combinations of treatments and controls. This also isn't very interesting because of the lack of clear differences between populations.

You can see the raw data here.

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