SAFS- Roberts Lab
McCartha Hintz Fowler Axton and Jenn
01/15/16 Running standard curve on C. gigas
Created by Michelle McCartha
Goal:
1-To train the students in making and running reactions for qPCR
2- To run standards curve.
Methods:
Aliquoting primers and probe
Probes and primers were aliquoted into 100Microliter 10micromolar concentrations using molecular water.
Each student was responsible for aliquoting one of the primers or 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.
*Need to start making premade aliquots of the primers/probe so that we don’t have to keep freezing and thawing them.
Preparing master mix
Master mix for C. gigas was prepared by Michelle to save on time but was explained as we went.
Mix was prepared by first adding the IQ Powermix, then FWD/REV primers and lastly probe.
*Brent came to visit during plate preparation and advised on how to make the reactions more precise and accurate. We can do this by adding water to the mix as it should be added. We will incorporate this step into future methods.
The following volumes comprised the master mix.
| 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 |
Plate set up
When finished the students finished thawing out the samples for the standard curve. All samples are using 10 day old C gigas larvae in increments of either 1, 5, 10, 25, 50. There are three standard curves to run and these “bio” reps will be run in triplicate. Ashley will set up the plate for all three technical reps for bio 1 curve, Jenn will set up the reps for Bio 2 curve and Axton will set up the reactions for the Bio 3 curve.
The plate was set up as follows:
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | |
| A | NTC | 1PCg5 | 1PCg1 | 1PCg1 | 2Cg1 | 2Cg1 | 2Cg1 | 3Cg1 | 3Cg1 | 3Cg1 | ||
| B | NTC | 1Cg1 | 1Cg5 | 1Cg5 | 2Cg5 | 2Cg5 | 2Cg5 | 3Cg5 | 3Cg5 | 3Cg5 | ||
| C | NTC | 1Cg10 | 1Cg10 | 1Cg10 | 2Cg10 | 2Cg10 | 2Cg10 | 3Cg10 | 3Cg10 | 3Cg10 | ||
| D | NTC | 1Cg25 | 1Cg25 | 1Cg25 | 2Cg25 | 2Cg25 | 2Cg25 | 3Cg25 | 3Cg25 | 3Cg25 | ||
| E | TC | 1Cg50 | 1Cg50 | 1Cg50 | 2Cg50 | 2Cg50 | 2Cg50 | 3Cg50 | 3Cg50 | 3Cg50 | ||
| F | ||||||||||||
| G | ||||||||||||
| H |
Sample reactions were made by first adding 29μL of the mix, followed by 4μL of the template and finally 17μL of molecular water.
NTC was made by adding 29μL of mix and 21μL of water
TC was made by adding 29μL of the mix, followed by 4μL of the template and finally 17μL of molecular water. Template for TC was from 25 18 day old C. gigas larvae.
There was only one mix up which was on A1 and A2. These standards were flipped.
When the students were finished with the plate, the lids were double checked that they were capped and then the plate was centrifuged for 1 min at 2000RPM.
Running qPCR
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 tad file as 20160115_144909
Results
All standard curve data is attached including for this curve.
Roberts Lab 