SAFS- Roberts Lab
12/10/15 Oly and Pg Standard Curve 1
Created by: Michelle McCartha
Goal: Want to run the OLY and Pg standard curves today to test curve and then will run again for repeatability tomorrow.
Aliquoting primers and probe
(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)|
Plate setup will as specified below.
For all reactions, the mix was placed in the well first followed by the template/standard/water (NTC)
TC was made up of 25 larvae
For all reactions volume was brought up to 50 microliters with water- water was not added to the master mix directly.
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. 20151211_143614
All standard curve data is in the attached excel file including from this run.