SAFS- Roberts Lab
McCartha
02/08/16 Running other two size classes of C. gigas for comparison
Created by: Michelle McCartha
Goal:
In literature, a difference in amplification has been noted when comparing different size classes of the same species using qPCR due to sensitivity and there being more genetic material at different stages of species maturity. To investigate this further and confirm if or not we need to consider size fractions of our species when processing samples, a test will be done using the three age classes of C. gigas that we have in stock. These size classes include 18-day old, 10-day old and 3-day old larvae. We currently have three bio reps prepared and already digested using te ProK method that we have been using. We already have a curve for the 10-day old age class, so today only the 3-day old and the 18-day old will be run using all three sets of standard curves in triplicate.
Methods:
Aliquoting primers and probe
C1V1=C2V2
(100µM)(x)=(10µM)(200µL)x=20µL of each primer
200µL aliquot-10µL primer=180µL 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
Only one master mix needs to be prepared for this set since we are working with the same species.
The reactions will be run using the same 50 microliter volume as done with the previous C. gigas curve for consistency.
Mix was made by adding the IQpowermix, followed by FWD/REV primers and probe and lastly with water.
The mix was made in a 15ml sterile centrifuge tube.
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 | 100 | 2500 | 250 | 2750 | 2750 |
| FWD Primer | 1.5 | 100 | 150 | 15 | 165 | 165 |
| Rev Primer | 1.5 | 100 | 150 | 15 | 165 | 165 |
| Probe | 1 | 100 | 100 | 10 | 110 | 110 |
| Water | 17 | 100 | 1700 | 170 | 1870 | 1870 |
Preparing Plate
Two plates will be run- one for the 18-day old and one for the 3-day old larvae age classes. Even thought they will have different plates, the plates will be set up the same for each run as specified below.
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | |
| A | NTC | 1Cg1 | 1Cg1 | 1Cg1 | 2Cg1 | 2Cg1 | 2Cg1 | 3Cg1 | 3Cg1 | 3Cg1 |
| B | NTC | 1Cg5 | 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 |
For al reactions, the mix was placed in the well first followed by the template/standard/water (NTC)
TC was made up of 25 18-day or 3-day old larvae respective to the plate standards and was pulled from Bio 1 reps.
qPCR parameters
Changed volume reaction on methods to 50 microliters.
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
18-day old age class- Saved tad file as 20160208_142850
3-day old age class- Saved tad file as 20160208_122545
Results
Excel file attached includes all standard curve data for all species including the runs performed here.
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