SAFS- Roberts Lab
McCartha
02/05/16 Running Vp standard curve
Created by Michelle McCartha
Goal: To test complete set of the standard curve and determine if the primers and probe and curve are ready for running samples.
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.
Master mix prep
Took out samples and allowed to thaw while making master mix.
To make the master mix, the following volumes were used…including water in 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 |
| Water | 17 | 50 | 850 | 85 | 935 | 935 |
Water was added to the mix in accordance with the new method protocol.
To make the master mix, a 15ml centrifuge tube was used. The IQ Powermix was added first, followed by the FWD/REV primers and probe and finally the molecular water.
The mix was finger vortexed as well as pipetted up and down to allow homogeneity.
Plate Set up
Set up for the plate is as follows:
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | |
| A | NTC | 1Vp1 | 1Vp1 | 1Vp1 | 2Vp1 | 2Vp1 | 2Vp1 | 3Vp1 | 3Vp1 | 3Vp1 | |
| B | NTC | 1Vp5 | 1Vp5 | 1Vp5 | 2Vp5 | 2Vp5 | 2Vp5 | 3Vp5 | 3Vp5 | 3Vp5 | |
| C | NTC | 1Vp10 | 1Vp10 | 1Vp10 | 2Vp10 | 2Vp10 | 2Vp10 | 3Vp10 | 3Vp10 | 3Vp10 | |
| D | NTC | 1Vp25 | 1Vp25 | 1Vp25 | 2Vp25 | 2Vp25 | 2Vp25 | 3Vp25 | 3Vp25 | 3Vp25 | |
| E | TC | 1Vp50 | 1Vp50 | 1Vp50 | 2Vp50 | 2Vp50 | 2Vp50 | 3Vp50 | 3Vp50 | 3Vp50 | |
| F | |||||||||||
| G | |||||||||||
| H |
Sample reactions were made by adding 46μL of the master mix followed by 4μL of the template.
NTC was made by adding 46μL of master mix followed by 4μL of water
TC was made using 25 18-day manilla clams digested in 1ml Prok solution as all samples were digested.
TC reactions were made by adding 46μL of the master mic followed by 4μL of the template.
When all the reactions were prepped, checked to make sure the caps were on securely and centrifuged for 1 min at 2000RPM.
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 tad file as 20160205_150807
Results:
Manila clam standard curve data is in the attached excel file.
