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:

CurrentStandardCurveData

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

ResultsCurrentStandardCurveData

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.

CurrentStandardCurveData

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.

CurrentStandardCurveData

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.

Comparison of changing volume…
55.7 KBComparison of changing volume reaction 50-25

Evan’s Capstone – eNotebook 5

Wednesday 2/10/2016 and Thursday 2/11/2016

            Double update today since I didn’t have time to write up my report yesterday night and figured it would be simple to fill it in here! Wednesday was relatively simple, just finished up the final 7 RNA Isolations while today I worked on learning how to conduct proper DNAsing of the samples to prepare for reverse transcription and then qPCR.

Wednesday’s RNA Isolations:

3BRC and 9BRC (Rock Creek) in addition to 7BCO and 17BCO (Coulter Creek) were pulled at 12:20 PM to be used as the first set. The second set consisted of 1BRC and 12BRC with 19BCO and were pulled at 12:55PM. Samples were returned to the -80 freezer at 4:05 PM.

Only one slight deviation from the standard procedure that day and it was that Set 1 rested on ice after step 15 for 10 minutes while waiting for Set 2 in the centrifuge.

Nanodrop Results:

Example: X-B-SampleSite – 260/280: ###, 260/230: ###, Concentration ### ng/uL
1BRC – 1.82, 1.01, 436.4
3BRC – 1.95, 2.20, 1393.7
9BRC – 1.97, 2.14, 1279.0
12BRC – 1.91, 2.20, 872.8
7BCO – 1.92, 2.05, 611.0
17BCO – 1.92, 2.08, 766.1
19BCO – 1.91, 2.28, 925.0

Thursday’s DNAsing:

Relatively simple to learn, it went smoothly as far as I can tell other than some of the samples having lower post-DNAse nanodrops than before. The procedure was the same as what is listed in the Common Lab Protocols except on the step where on step where you add the deactivation reagent and incubate it, we let it incubate for 5 minutes as per the manufacturer procedure.

The first set of isolated samples was pulled at 12:05 PM and consisted of 3BMA and 5BMA in addition to 13BHA and 14BHA. As a note, 14BHA was the sample that only had 25 uL total of isolated sample because of a small liver sample size. This led me to adapt a procedure and dilute it down to 40 ng/uL as compared to the normal 200 ng/uL for every other sample concentration since its initial concentration was only 180 (with only about 20 uL when it calls for about 44). The second set of samples consisted of 6BRC and 7BRC (Rock Creek) with 11BSW and 13BSW (Swamp Creek) and were pulled at 2:05 PM.

Nanodrop Results Post-DNAse (same format):
3BMA – 1.97, 1.47, 109.6
5BMA – 1.96, 1.32, 125.8
13BHA – 2.01, 1.75, 111.1
14BHA – 1.89, 1.04, 28.3
6BRC – 1.97, 1.68, 107.8
7BRC – 1.99, 1.60, 111.8
11BSW – 1.91, 1.43, 113.5
13BSW – 1.98, 1.55, 130.3

Samples were returned to the -80 freezer in a new storage container (to signify they were DNAsed) at 3:55 PM for Set 1 and 5:30 PM for Set 2. It appears that a lot of concentration (and 260/230 values) were lost in the process of DNAsing, given that the 260/230 numbers normally decrease with concentration this was expected and given how little actual RNA is in 14BHA I am not surprised it only has a value of 1.04. The other samples were okay, was really wanting more in the 1.60 range but the 1.4+’s are okay too. Most of the samples lost a lot of concentration to DNAsing which was also expected since liver tissue samples are normally pretty high in DNA and RNA and then by removing the DNA it would make sense for the concentration to dip down more than other types of samples. Overall a good learning experience and the samples are likely still viable. Next day in lab will likely be better and I should be able to get all the other samples done if I work efficiently!