DNR Geoduck Project- Michelle McCartha

SAFS- Roberts Lab
McCartha
12/10/15 Oly and Pg Standard Curve 1
Created by: Michelle McCartha
Goal: Want to run the OLY and Pg standard curves today to test curve and then will run again for repeatability tomorrow.
Methods:
Aliquoting primers and 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.

Took out all samples to be thawed out. Used hand friction to assist thawing as necessary.

Preparing master mix
Mix was made by adding the IQpowermix, followed by FWD/REV primers and probe.
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 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
Preparing Plate
Plate setup will as specified below.
1 2 3 4 5 6 7 8 9 10
A NTC 1Oly1 1Oly1 1Oly1 2Oly1 2Oly1 2Oly1 3Oly1 3Oly1 3Oly1
B NTC 1Oly5 1Oly5 1Oly5 2Oly5 2Oly5 2Oly5 3Oly5 3Oly5 3Oly5
C NTC 1Oly10 1Oly10 1Oly10 2Oly10 2Oly10 2Oly10 3Oly10 3Oly10 3Oly10
D NTC 1Oly25 1Oly25 1Oly25 2Oly25 2Oly25 2Oly25 3Oly25 3Oly25 3Oly25
E TC 1Oly50 1Oly50 1Oly50 2Oly50 2Oly50 2Oly50 3Oly50 3Oly50 3Oly50
F
G
H
For all reactions, the mix was placed in the well first followed by the template/standard/water (NTC)
TC was made up of 25 larvae
For all reactions volume was brought up to 50 microliters with water- water was not added to the master mix directly.
 
qPCR parameters
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 file as tad. 20151211_143614
Results
All standard curve data is in the attached excel file including from this run.

DNR Geoduck Project- Michelle McCartha

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.

Cg_SC_age10_20160115_144909

Cg_SC_age18_20160208_142850

DNR Geoduck Project-Michelle McCartha

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.

1/28/16 Re-do geoduck curve with proper technique for master mix

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