SAFS-Roberts Lab
McCartha
1-29-16- Re-run geoduck standard curve to test for machine precision
Created by Michelle McCartha
Goal: to determine if a standard curve should be run on every plate and on each day of the run, we want to test the precision of the qPCR machine. To do this the exact same methods will be used as were used on 1/28/16 when the original curve was run. This data will then be compared.
Methods:
Geoduck samples we set out to thaw.
Probes and primers were aliquoted into 100Microliter 10micromolar concentrations using molecular water.
10microliter primer and probe with 90 microliter molecular water respectively. These were mixed using pipette and centrifuged down.
Prepared master mix using a sterile 15ml centrifuge tube using volumes calculated below.
| 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 |
Added super mix first then primers and probe followed by water.
Finished Thawing standard curve samples by hand friction.
Pulled clean 96-well plate and caps. caped all rows that we are going to use.
Prepared plate by adding sample first then topping off with mix.
Continuing with 50 microliter reactions so added 4 microliters sample and 46 microliters master mix.
Prepare NTC first by adding 46 microliters master mix following the 4 microliter water for no template.
Capped these reactions before moving onto samples and template control.
Template control was added by adding 4 microliters template control adn 46 microliters master mix.
Lay out for all reactions below.
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | |
| A | NTC | 1Pg1 | 1Pg1 | 1Pg1 | 2Pg1 | 2Pg1 | 2Pg1 | 3Pg1 | 3Pg1 | 3Pg1 |
| B | NTC | 1Pg5 | 1Pg5 | 1Pg5 | 2Pg5 | 2Pg5 | 2Pg5 | 3Pg5 | 3Pg5 | 3Pg5 |
| C | NTC | 1Pg10 | 1Pg10 | 1Pg10 | 2Pg10 | 2Pg10 | 2Pg10 | 3Pg10 | 3Pg10 | 3Pg10 |
| D | NTC | 1Pg25 | 1Pg25 | 1Pg25 | 2Pg25 | 2Pg25 | 2Pg25 | 3Pg25 | 3Pg25 | 3Pg25 |
| E | TC | 1Pg50 | 1Pg50 | 1Pg50 | 2Pg50 | 2Pg50 | 2Pg50 | 3Pg50 | 3Pg50 | 3Pg50 |
| F | ||||||||||
| G | ||||||||||
| H |
When all reactions were prepared, centrifuged for 1 min at 2000RPM.
Took to qPCR machine and ran curve under the following program parameters.
Step 1) 95.0°C-10 minutes
Step 2) 95.0°C-20 seconds
Step 3) 65°C-20 seconds
Step 4) 72°C-30 seconds
Step 5) Repeat steps 2-4 39 more times (40 times total)
Step 6) 72°C-2 minutes
Step 7) Hold at 4°C forever
Saved file as tad file: 20160129_124635
Results
All standard curve data including for this one is attached.
Roberts Lab 