Ran qPCRs on the O.lurida total RNA I isolated on 20150506 to assess presence of gDNA carryover with Oly Actin primers (SR IDs: 1505, 1504).

Used 1μL from all templates.

All samples were run in duplicate.

Positive control was HL1 O.lurida DNA isolated by Jake on 20150323.

Master mix calcs are here: 20150512_qPCR_Oly_RNA

Cycling params:

• 95C - 3mins

• 40 cycles of:

  • 95C - 5s

  • 60C - 20s

• Melt curve

Plate layout: 20150512_qPCR_plate_Jake_Oly_HS_RNA

Results:

qPCR Data File (Opticon2): Sam_20150512_123246.tad

qPCR Report (Google Spreadsheet):20150512_qPCR_Report_Jake_Oly_HS_RNA

Excluding the no template controls (NTC), all samples produced amplification. Will require DNasing before making cDNA.

Related to the qPCR I ran earlier today with these same primers, the efficiencies of the reactions on this plate are significantly better (i.e. normal; >80% efficiencies) than the earlier qPCR. The improved efficiency would also explain why the positive control comes up two cycles earlier on this run.

In the amplification plots below, the positive control reps are the two lines coming up at cycle ~20.

Amplification Plots

(http://eagle.fish.washington.edu/Arabidopsis/20150512qPCR_Amp_Jake_Oly_HS_RNA.JPG)

Melt Curves

(http://eagle.fish.washington.edu/Arabidopsis/20150512qPCR_Melt_Jake_Oly_HS_RNA.JPG)