DNR Geoduck Project- Michelle McCartha

SAFS- Roberts Lab
McCartha
12/10/15 Oly Standard Curve 1
Created by: Michelle McCartha
Goal: Want to run the OLY standard curve today to test curve and then will run again for repeatability tomorrow.
Methods:
Aliquoting primers and probe
C1V1=C2V2
     (100µM)(x)=(10µM)(100µL)x=10µL of each primer 
     100µL aliquot-10µL primer=90µL of nuclease free water
Added water then Primer stock solution and pipetted up and down to mix.
Inverted tubes and finger vortexed then centrifuged.

Took out all samples to be thawed out. Used hand friction to assist thawing as necessary.

Preparing master mix
Mix was made by adding the IQpowermix, followed by FWD/REV primers and probe.
Once all components of the mix was added, the tube was inverted and liquid pipetted up and down for mixing.
Master Mix Solutions Standard volume (μL) Multiply By new volume * 10% pipette error add  pipette error Final volume to add (μL)
Master mix 25 50 1250 125 1375 1375
FWD Primer 1.5 50 75 7.5 82.5 82.5
Rev Primer 1.5 50 75 7.5 82.5 82.5
Probe 1 50 50 5 55 55
Preparing Plate
Plate setup will as specified below.
1 2 3 4 5 6 7 8 9 10
A NTC 1Oly1 1Oly1 1Oly1 2Oly1 2Oly1 2Oly1 3Oly1 3Oly1 3Oly1
B NTC 1Oly5 1Oly5 1Oly5 2Oly5 2Oly5 2Oly5 3Oly5 3Oly5 3Oly5
C NTC 1Oly10 1Oly10 1Oly10 2Oly10 2Oly10 2Oly10 3Oly10 3Oly10 3Oly10
D NTC 1Oly25 1Oly25 1Oly25 2Oly25 2Oly25 2Oly25 3Oly25 3Oly25 3Oly25
E TC 1Oly50 1Oly50 1Oly50 2Oly50 2Oly50 2Oly50 3Oly50 3Oly50 3Oly50
F
G
H
For all reactions, the mix was placed in the well first followed by the template/standard/water (NTC)
For all reactions, volume was brought to 50 microliters with water at the plate stage, not mixed in the master mix directly.
TC was made up of 25 larvae
 
qPCR parameters
Plate was run at the following parameters:
    • Incubate 95C for 2 minutes 30 seconds
    • Incubate 95C for 30 seconds
    • Incubate 60C for 50 seconds
    • Plate read
    • Go to step 2, 39 more times
Saved file as tad 20151210_130740
Results and data on the Oly 1 standard curve are in the attached excel file.
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