SAFS- Roberts Lab
McCartha
1/28/16 Re-do geoduck curve with proper technique for master mix
Created by McCartha
Goal- Previously, the master mix was prepared with out adding water to the mix, instead adding water to each individual reaction. This may have effected the resulting curve during qPCR. Today, the mix will be prepared using the correct technique and we should see greater replicate precision.
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: 20160128_142551
Results
Data file attached has results from all curves including this one.
Roberts Lab 