SAFS- Roberts Lab
McCartha
02/05/16 Running all species standard curves with 25 microliter volume reactions
Created by: Michelle McCartha
Goal: Micah wants us to start running all reactions at a 25 microliter volume reaction instead of the current 50 microliter volume. To test the 25 microliter volume on all species, bio rep 1 standard curve will be run using the new volume to test reproducibility of data with the changed volume.
Methods
Primer and probe aliquots
No new aliquots of primers and probe were necessary as we had some left over from previous curves.
Master mix
One master mix per species was made using the following volumes of materials.
| Master Mix Solutions | Standard volume (μL) | Multiply By | new volume | * 10% pipette error | add pipette error | Final volume to add (μL) |
| Master mix | 12.5 | 18 | 225 | 22.5 | 247.5 | 247.5 |
| FWD Primer | 0.75 | 18 | 13.5 | 1.35 | 14.85 | 14.85 |
| Rev Primer | 0.75 | 18 | 13.5 | 1.35 | 14.85 | 14.85 |
| Probe | 0.5 | 18 | 9 | 0.9 | 9.9 | 9.9 |
| Water | 10.5 | 18 | 189 | 18.9 | 207.9 | 207.9 |
When the master mix was made, they were inverted for mixing and spun down briefly.
The samples were set out to thaw with assistance using hand friction
The 10-day old C gigas samples were used so that they could be compared with current curve data.
When all samples were thawed, they were finger vortexed for mixing and spun down briefly.
Plate set up
The 96-well PCR plate was set up using the master mix for each species and the Bio rep 1 standard curve samples.
23microliters of the master mix was added first followed by 2 microliters of the samples.
For the NTC 2 microliters replaced the Bio rep 1 sample volume
For the TC DNA from digested TC containing DNA from 25 larvae of each species (independently) was used. There were 4 template control digestions, 1 for each species.
The plate was spun down at 2000RPM for 1 minute.
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | |
| A | NTC | NTC | NTC | NTC | ||||||||
| B | NTC | NTC | NTC | NTC | ||||||||
| C | TC | TC | TC | TC | ||||||||
| D | 1Pg1 | 1Pg1 | 1Pg1 | 1Vp1 | 1Vp1 | 1Vp1 | 1Oly1 | 1Oly1 | 1Oly1 | 1Cg1 | 1Cg1 | 1Cg1 |
| E | 1Pg5 | 1Pg5 | 1Pg5 | 1Vp5 | 1Vp5 | 1Vp5 | 1Oly5 | 1Oly5 | 1Oly5 | 1Cg5 | 1Cg5 | 1Cg5 |
| F | 1Pg10 | 1Pg10 | 1Pg10 | 1Vp10 | 1Vp10 | 1Vp10 | 1Oly10 | 1Oly10 | 1Oly10 | 1Cg10 | 1Cg10 | 1Cg10 |
| G | 1Pg25 | 1Pg25 | 1Pg25 | 1Vp25 | 1Vp25 | 1Vp25 | 1Oly25 | 1Oly25 | 1Oly25 | 1Cg25 | 1Cg25 | 1Cg25 |
| H | 1Pg50 | 1Pg50 | 1Pg50 | 1Vp50 | 1Vp50 | 1Vp50 | 1Oly50 | 1Oly50 | 1Oly50 | 1Cg50 | 1Cg50 | 1Cg50 |
qPCR parameters
Changed volume reaction on methods to 25 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
Saved tad file as 20160205_180258
Results
For some reason no data for HEX was collected during the run. Need to see if there is something that I need to do about seeing the data. FAM data was collected though and so the screen shot is for the Oly and the Gigas runs. The excel file graphs the comparisons for Bio Rep 1 on both species for both reaction volumes. There is a difference in the results that may need further testing.
Comparison of changing volume…
