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

Evan’s Capstone – eNotebook 3

Thursday – 1/28/2016

Plan for today changed slightly, just continuing more RNA isolations in the hopes of getting them all done by next week and continuing on to the next part of the project. Operation running smoothly now, essentially had no downtime during sample work with the centrifuge running with either set of samples at a time. Also started work on two new creeks now that the first 6 samples of Swamp Creek and May Creek are finished!

First set of samples consisted of 11 and 13 of Issaquah (#BIS) in addition to 13 and 14 of Harris (#BHA) and were pulled at 11:50 AM. The second set was 12 and 15 of Harris with 3 and 17 of Issaquah which were pulled at 12:10 PM.

A few more deviations this time due to balancing centrifuge time with my supervisor; Set 1 rested for 4 additional minutes after Step 18 and then after Step 21 rested for longer than anticipated (8 minutes) and was re-centrifuged for 3 minutes to ensure integrity of pellet. Set 2 rested after Step 13 for 4 additional minutes and also rested after Step 18 for 2 additional minutes. Sample 14BHA ended up being re-suspended in 25 ul of DEPC-treated water instead of the usual 50 because of a small liver sample and subsequently a small pellet size.

Nanodrop results for the day:

12BHA – 260/280: 1.97, 260/230: 2.01, Concentration 720.6
13BHA – 1.98, 2.26, 1574.7
14BHA – 1.74, 2.08, 391.0 (~)
15BHA – 1.94, 2.09, 774.5

3BIS – 1.95, 2.09, 844.6
11BIS – 1.96, 1.90, 644.8
13BIS – 1.93, 2.27, 977.6
17BIS – 1.82, 1.57, 241.1 (~)

The tilde is to represent the two samples I am questioning on using given the less than optimal numbers (both have numbers lower than 1.8 which is what I’ve been looking for). In hindsight, 17BIS had a small liver sample size similar to 14BHA and given how low the concentration is, it probably would have been smart to limit the re-suspension to 25 ul for it also. I’m not sure if the small liver sample size is to blame for the low Nanodrop numbers? I will need to consult with someone about what to do with them.

All samples were placed in the -80 freezer at 4:40 PM following the Nanodrop results. My goal for next week will be to get at least 6 samples from each creek finished so that they can be brought through the next steps and start to see some results. May or may not have to pull an extra sample for Harris and Issaquah given the two “maybes” from today; We’ll see!

#rna, #salmon

Evan’s Capstone – eNotebook 2

Tuesday – 1/26/2016

Coho Salmon and Cutthroat Trout Liver Analyses

Continuing work on RNA isolations today, managed to double up on the samples run per day and actually ended up easier to do than Thursday’s due to running homogenization during incubation steps.

Samples were pulled from Swamp Creek and May Creek again, Set 1 being samples 2May, 6May, 9Swamp, 14Swamp at 11:36 AM; Set 2 consisted of 1May, 7May, 6Swamp, and 18Swamp and were pulled at 12:54 PM.

Samples were ran through RNA Isolation as indicated in the Common Lab Protocols like last Thursday’s samples (found at https://github.com/sr320/LabDocs/wiki/Common-Lab-Protocols).

Some more minor deviations due to timing between sets, less than before given overlap went pretty well overall during incubation times. The 1^st set of samples were kept on ice from 2:30 PM to 3:30 PM until time was available to do the Nanodrop. The 2^nd set of samples had to sit at Step 13 for 2 extra minutes, and on Step 10 for 3 minutes, then before Step 20 they had to remain in the isopropanol for 10 minutes while waiting for Nanodrops for the 1^st set.

Nanodrop samples came back better than before (likely due to being more comfortable with the machine) and were conducted the same way as Thursday’s samples.

Set 1 ~
2BMA – 260/280: 1.99; 260/230: 2.14; Concentration: 1466.4
6BMA – 1.84, 1.87, 422.4
9BSW – 1.98, 2.12, 1505.1
14BSW – 1.98, 1.86, 1387.5

Set 2 ~
1BMA – 1.96, 1.82, 889.9
7BMA – 2.00, 2.10, 1841.1
6BSW – 1.98, 2.14, 1410.4
18BSW – 1.99, 1.82, 1699.6

Samples in Set 1 were placed in the -80 Celsius freezer at 3:50 PM while the Set 2 samples were in the freezer by 4:50 PM.

Seems to be even better than last week, plan for Thursday is to learn the process of DNasing the RNA samples and likely more RNA isolations!

Why can I not create…

Why can I not create a list?? It cannot be that hard.

Evan’s Capstone – eNotebook 1

Thursday – 1/21/2016

First day of Lab work on samples! Excited to get started on the study sites for the capstone project.

Samples from Swamp Creek (11 and 13) and May Creek (3 and 5) were pulled at 12:50 to be taken through the process of RNA Isolation as outlined in the Common Lab Protocols.

Some minor deviations due to conducting two different “sets” of two samples at a time and some overlaps when using the centrifuge with another lab member. Maybe try to figure out a better method of conducting multiple samples, maybe sets of 4 and then during the first incubation/centrifuging step set up another set of 4 samples?

During the first incubation of 15 minutes the second set of samples (5 and 13) incubated for 19 minutes while the first set of samples were in the centrifuge. After the 750 ul supernatant was pulled off and placed in a new snap-cap tube these two samples also rested for 5 minutes while waiting on the final ethanol washing step for the first set of samples (3 and 11). The first set of samples also had to be set to rest for 12 minutes before the final ethanol centrifuge, the ethanol supernatant was pulled off and the sample was set capped to ensure the ethanol did not evaporate off completely before the second 400 ul of ethanol could be added and the final washing step could be conducted.

The first set of samples were capped and held in the normal refrigerator until the second set was finished so we could bring them to the Nanodrop machine.

We set up the machine by first cleaning off the upper and lower lenses, placing 1 ul of clean water and initializing the nucleic acid option then cleaning the lenses and repeating the step again choosing the blank option. The lenses were cleaned in between each 1 ul sample and using the “measure” option.

Some errors occurred with new use of the machine (lower values or higher concentrations than expected, air bubbles in some situations, and some material not fully suspended on another sample), however the values from the successful runs of the machine were collected:

(ex. 3BMA is 3rd Sample, B for Cutthroat, MA for May Creek, SW for Swamp Creek)
3BMA – 260/280: 1.99; 260/230: 1.92; Concentration ng/ul: 1291.1
5BMA – 1.97, 1.80, 1111.3
11BSW – 1.96, 1.80, 745.7
13BSW – 1.98, 1.92, 1221.1

Completed samples placed back into the container and stored in -80 Celsius freezer at 5:15 PM and work concluded.

Good results and data collected so far and ready for work on Tuesday!

#rna, #salmon