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