DNR Geoduck Project: Michelle McCartha

SAFS- Roberts Lab
McCartha Hintz Fowler Axton and Jenn
01/15/16 Running standard curve on C. gigas
Created by Michelle McCartha
Goal:
1-To train the students in making and running reactions for qPCR
2- To run standards curve.
Methods:
Aliquoting primers and probe
Probes and primers were aliquoted into 100Microliter 10micromolar concentrations using molecular water.
Each student was responsible for aliquoting one of the primers or 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.
*Need to start making premade aliquots of the primers/probe so that we don’t have to keep freezing and thawing them.

Preparing master mix

Master mix for C. gigas was prepared by Michelle to save on time but was explained as we went.
Mix was prepared by first adding the IQ Powermix, then FWD/REV primers and lastly probe.
*Brent came to visit during plate preparation and advised on how to make the reactions more precise and accurate. We can do this by adding water to the mix as it should be added. We will incorporate this step into future methods.
The following volumes comprised 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
Plate set up
When finished the students finished thawing out the samples for the standard curve. All samples are using 10 day old C gigas larvae in increments of either 1, 5, 10, 25, 50. There are three standard curves to run and these “bio” reps will be run in triplicate. Ashley will set up the plate for all three technical reps for bio 1 curve, Jenn will set up the reps for Bio 2 curve and Axton will set up the reactions for the Bio 3 curve.
The plate was set up as follows:
1 2 3 4 5 6 7 8 9 10 11 12
A NTC 1PCg5 1PCg1 1PCg1 2Cg1 2Cg1 2Cg1 3Cg1 3Cg1 3Cg1
B NTC 1Cg1 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
Sample reactions were made by first adding 29μL of the mix, followed by 4μL of the template and finally 17μL of molecular water.
NTC was made by adding 29μL of mix and 21μL of water
TC was made by adding 29μL of the mix, followed by 4μL of the template and finally 17μL of molecular water. Template for TC was from 25 18 day old C. gigas larvae.
There was only one mix up which was on A1 and A2. These standards were flipped.
When the students were finished with the plate, the lids were double checked that they were capped and then the plate was centrifuged for 1 min at 2000RPM.
Running qPCR
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 20160115_144909

Results
All standard curve data is attached including for this curve.

DNR Geoduck Project: Michelle McCartha

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.

DNR Geoduck Project: Michelle McCartha

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.