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.
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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.

DNR Geoduck Project- Michelle McCartha

SAFS- Roberts Lab
McCartha
02/05/16 Running Vp standard curve
Created by Michelle McCartha
Goal: To test complete set of the standard curve and determine if the primers and probe and curve are ready for running samples.
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.
 
Master mix prep
Took out samples and allowed to thaw while making master mix. 
To make the master mix, the following volumes were used…including water in 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
Water 17 50 850 85 935 935
 
Water was added to the mix in accordance with the new method protocol.
To make the master mix, a 15ml centrifuge tube was used. The IQ Powermix was added first, followed by the FWD/REV primers and probe and finally the molecular water. 
The mix was finger vortexed as well as pipetted up and down to allow homogeneity. 
 
Plate Set up 
Set up for the plate is as follows:
 
1 2 3 4 5 6 7 8 9 10 11
A NTC 1Vp1 1Vp1 1Vp1 2Vp1 2Vp1 2Vp1 3Vp1 3Vp1 3Vp1
B NTC 1Vp5 1Vp5 1Vp5 2Vp5 2Vp5 2Vp5 3Vp5 3Vp5 3Vp5
C NTC 1Vp10 1Vp10 1Vp10 2Vp10 2Vp10 2Vp10 3Vp10 3Vp10 3Vp10
D NTC 1Vp25 1Vp25 1Vp25 2Vp25 2Vp25 2Vp25 3Vp25 3Vp25 3Vp25
E TC 1Vp50 1Vp50 1Vp50 2Vp50 2Vp50 2Vp50 3Vp50 3Vp50 3Vp50
F
G
H
Sample reactions were made by adding 46μL of the master mix followed by 4μL of the template.  
NTC was made by adding 46μL of master mix followed by 4μL of water
TC was made using 25 18-day manilla clams digested in 1ml Prok solution as all samples were digested. 
TC reactions were made by adding 46μL of the master mic followed by 4μL of the template. 
When all the reactions were prepped, checked to make sure the caps were on securely and centrifuged for 1 min at 2000RPM.
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 tad file as 20160205_150807

Results:

Manila clam standard curve data is in the attached excel file.
 
 
 
 

DNR Geoduck Project- Michelle McCartha

SAFS- Roberts Lab
McCartha
12/10/15 Oly Standard Curve 1
Created by: Michelle McCartha
Goal: Want to run the OLY standard curve 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)
For all reactions, volume was brought to 50 microliters with water at the plate stage, not mixed in the master mix directly.
TC was made up of 25 larvae
 
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 20151210_130740
Results and data on the Oly 1 standard curve are in the attached excel file.

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