Laura’s Notebook: Mid-August, 2019 goals & timeline

QuantSeq:

  • By August 20th, finish prepping RNA samples for QuantSeq library prep. This includes:
    • Re-running qPCR on a subset of samples with the Actin primer set that detects gDNA.
    • Quantifying remaining RNA with Qubit
    • Concentrate dilute samples. Goal is 500 ng in 5 uL.
    • Assess RNA quality via Bioanalyzer on subset of samples.
  • Week of August 19th:
    • Get all materials ready for QuantSeq library prep.
    • Test protocol on 8 samples (“extra” samples).
  • Week of Aug. 26th, if all materials are in lab: begin library prep!

Degree:

  • Identify and reach out to potential new committee members
  • Read Jackie’s stack of papers, take notes and organize using Evernote
  • Get study lists from Rick & Steven

Manuscripts:

  • Finish simplifying O. lurida temperature/food methods, results and discussion. Get feedback from Steven.
  • Begin revising, or at least organize thoguhts on, Polydora MS as per discussion with Chelsea & Julieta.

PCSGA:

  • Review presentation from Aquaculture 2019 for PCSGA.
  • Measure larvae from 2017 OA/T study, possibly include in PCSGA presentation? Maybe Kaitlyn can help with this. Maybe use Jackie’s fancy computer.

from The Shell Game https://ift.tt/2TzdCDN
via IFTTT

Laura’s Notebook: MACAU Run 1

I ran MACAU to assess the influence of Oly shell length on methylation counts, given relatedness. I used wet weight as a covariate. Check out this jupyter notebook for details.

Results, which are located in the https://ift.tt/2OSj8ma repo, in theanalyses/macau/ subdirectory, is a table with many columns. Thus far I have pulled the p-vlaues out to visualize a quick heatmap. I did this in the 04-raw-count-files.Rmd notebook (probably will move that elsewhere).

The heat map was generated by filtering the raw methylated count data with only loci that MACAU identified as DMLs, witht he very conservative p-value of 0.001. The samples mostly cluster by population, with the exception of samples 12 ans 18 – those are two South Sound samples (#10-18) that cluster with Hood Canal samples (#1-9).

image

from The Shell Game https://ift.tt/2yWuhYo
via IFTTT

Laura’s Notebook: Prepping for QuantSeq library prep

I am usig QuantSeq to look at gene expression in Oly larvae and adult ctenidia tissue, both collecting as part of the OA/Temp study. I have some preliminary data from the larvae which looks very interesting, suggests that adult exposure results in altered gene express in newly released larvae, despite larvae being fertilized and grown in common conditions. The preliminary run was 8 samples – 4 from treated parents, 4 from ambient parents – I’m sequencing a bunch more larvae from all treatments and cohorts to see if the parental treatment effects are consistent across populations. I will also detect gene expression differences among populations, which should also be interesting given their differing growth and survival rates.

QuantSeq, which is also known as “TagSeq” or “3’ mRNA-Seq”, sequences from the 3’ end of a transcript, and when building libraries it only generates one fragment per transcript, thus it should be highly accurate for gene expression studies. Library prep uses total RNA as input, and entails the following steps:

image

I spoke with Katherine to get some tips on this protocol. She says that it’s a beast, particularly since I have a ton of samples. She was extremely helpful, and in addition to tips via phone she wrote down important things in a google doc. I’ve pasted it here:

Tips

  • Concentrate samples if dilute
  • Randomize sample types between rows and lanes
  • 500 ng RNA initially (or at least 200), try to be consistent across samples but ok if not
  • Repeater pipette for ethanol
  • Magnetic plate that fits 96 well plate well
  • Make thermocycler protocols ahead of time. Make sure ramp speed is correct.
  • Make 250uL/sample 80% ethanol ahead of both purification steps
  • After library prep is done, do Quibit HS DNA on each library.
  • Use bioanalyzer HS DNA to verify library quality. This step can take a long time (and be kinda pricey), as it takes 45 min to run 10 samples. If your samples generally seem to all work, you can decide if you want to just spot check (across tissute types, batches, etc.). I recommend always doing bioanalyzer on samples that require >=16 cycles or had low input/quality DNA as these are more likely to fail
  • I will go through a TON of 200 uL pipette tips
  • Optimal # samples per batch is 40
  • Practice on 8 samples (extra samples) to get used to protocol
  • I will need foil or 96 well-plate tape. Always spin down plates before removing covering, to ensure no cross contamination.
  • During purification, make sure beads are all stuck to wall. If I accidentally suck some up into the pipette, put it back!
  • make fresh SYBR dilution: 1.25 uL 100x SYBR stock + 23.75 uL DMSO = 25 uL 5x SYBR
    https://ift.tt/306RWBD
    • Laura update: protocol says I need 2.5x working stock. Instead dilute 1.25 uL 100x SYBR into 48.75 uL DMSO, for 50 uL of 2.5x SYBR working solution.
  • Need to create a custom qPCR protocol for this, to determinet he # cycles to use for amplification. Samples will have varying # cycles! When calculating how many cycles, always round down (i.e. bad to over-cycle).
  • When doing PCR to amplify libraries, keeping track of sample location is hard. Be careful, plan accordingly.
  • To sequence, Katherine used a High Seq 4000, and did ~80 samples per lane. QUESTION: do technical replicates?
  • If sequencing facility has never done QuantSeq before, get protocol from Katherine (e.g. concentration to use).

Stuff to get

  • Lexogen PCR Add-on Kit for Illumina (Cat. No. 020).
  • SYBR green: umiprobe dsGreen for Real-Time PCR x100 (100 uL). https://ift.tt/306RWBD
  • Magnetic plate that works well with 96 well plate (PCR strips ok too)
  • 20-50 uL multichannel
  • 150 uL multichannel
  • To concentrate RNA: https://ift.tt/2Z3vUSN
  • Maybe more 200 uL pipette tips

To Do

  • I already submitted purchase requests for much of the “stuff to get”
  • Determine if we have appropriate magnetic plate. If not, borrow from Graham?
  • Work with Sam to get a few multichannel pipettes calibrated
  • Re-do qPCR batches III and IV
  • Quantify remaining RNA
  • Concentrate samples that need it
  • Check quality of RNA?
  • Test whole protocol with 8 “extra” samples

from The Shell Game https://ift.tt/2ZZ2cLS
via IFTTT

Laura’s Notebook: Testing more Oly primers for DNA contamination

The last two rounds of qPCR I did to test for DNA contamination (Rounds III & IV) had some odd results – melt curves showed some samples’ RFU to increase at high temp. I conferred with Sam and he agreed that I should re-do the last 2 runs. Also, suggested looking at the amplification curves. Turns out that none of my runs resulted in the positive control (my DNA from larvae) amplifying. Some points from Sam: “Regarding your lack of amplification of your positive control. This is most likely due to the presence of introns in the gDNA. I’m guessing the primers were designed off of mRNA, since they were being used to evaluate gene expression, and mRNA doesn’t have introns. The presence of introns in gDNA usually is a problem in qPCRs because the size of the gDNA that falls between the two primer annealing regions is too large to be amplified in the time frame used for qPCR. The solution is to find an existing primer set that is known to amplify gDNA during qPCR (amplicon size <200bp).”

One primer that Sam successfully used before is gone, so I’m testing 3 other primer sets that were ordered at the same time to find one that could work. To test, I’m running 3 positive controls, and 3 no template controls for each primer. I’m testing those outlined in blue, below:

Gone but worked in past:

  • 1505 Ol_Act_F
  • 1504 Ol_Act_R

Testin:

  • 1509 Ol_Ef1a_F
  • 1508 Ol_Ef1a_R
  • 1503 Ol_Arp_F
  • 1502 Ol_Arp_R
  • 1678 Flk_Actin_FWD
  • 1677 Flk_Actin_REV

For each of the primer sets , I diluted 1 uL of the FWD and REV into 9 uL, then created mastermixes using these calcs:

Date Test primer
# Samples 3
# Reactions 6
Template (RNA sample) (uL) 6.0
Sso Fast (uL) 66.0
Pf, 10 uM (uL) 3.3
Pr, 10 uM (uL) 3.3
DEPC-treated water (uL) 52.8
Total Volume in mastermix (uL) 125.4

Plate setup. I used DNA sample 2a as my positive controls.

8 9 10 11 12
A EFG1a + EFG1a + EFG1a + x x
B EFG1a – EFG1a – x x x
C x x x x x
D ARP + ARP + ARP + x x
E ARP – x x x x
F x ARP – ARP – x x
G Actin + Actin + Actin + x x
H Actin – Actin – Actin – x x

from The Shell Game https://ift.tt/2MXkUQt
via IFTTT

Laura’s Notebook: DNasing RNA, Round II-V

DNasing, Rounds II-V

I performed 3 rounds of DNase reactions (n=30 per round) on the Oly larval and ctenidia RNA. I followed the same protocol as Round 1, using 50 uL RNA for each sample, 5 uL buffer, 1 uL DNase, and 5 uL inactivation reagent.

There were two samples – 38 and 46 (extracted previously in 2018) – which had less than 50 uL total volume in the original RNA sample. For these I used 25 uL RNA, 2.5 uL buffer, 1 uL DNase and 2.5 uL inactivation reagent.

Several of the samples also required dilution prior to DNasing, since the maximum amount of RNA in a 50 uL reaction is 10 ug. For these I added 50 uL DEPC-treated water and vortexed to mix, prior to DNasing: 477, 43, 564, 291, 304, 306, 309, 315, 316, 317, 321, 325, 335, 345.

Some images of the Turbo DNase process

This is the Turbo DNase kit I used.

IMG_8846

In it is: A) Buffer B) TurboDNase enzyme C) Inactivation Reagent D) Ultrapure water (to resuspend inactivation reagent, if it has dried).

NOTE: the kit says it’s good for 50 reactions, but that’s for 100 uL volume / reaction. For me, the limiting factor was the DNase enzyme – there was 120 uL DNsae (used 1 uL per sample) – so I did ~110 samples with one kit before pulling DNase from the other.

IMG_8847

2 sets of labeled 0.5 mL tubes. Step 1 is to transfer 50 uL RNA into one set of tubes.

After adding buffer (10% of RNA volume, in my case 5 uL), and DNase (1 uL), I vortexted gently and incubated at 37C for ~25 minutes in the Thermo Cycler. It takes no time to heat up, keeps track of the time. Maximum # tubes at once is 30.

IMG_8851

IMG_8855

IMG_8852

I forgot to get images of the inactivation reagent step – the solution is milky white, and after mixing and incubating at rooom temp for 5 minutes, samples are centrifuged to “pellet” the reagent, then the supernatant containing RNA is transferred to the 2nd batch of tubes. NOTE: I held everything on ice while working.

qPCR to check for DNA contamination

I ran 3 more batches of qPCR to check for DNA contamination.

For these batches, I selected Olympia oyster primers ordered by Jake

Snip20190806_39

IMG_8853

Primers are located in the freezer in FTR 213.

IMG_8850

Found the Oly primers using the database.

image

I created working stocks of my primers – diluted 12 uL each primer in 108 uL DECP-treated water, creating 120 uL at 10 uM for each.

Created a mastermix for all reactions using the following calculations, then pipetted 19 uL mastermix onto qPCR plates, and 1 uL each sample in duplicate.

Date 1 plate , 32 rxns All 3 reactions
# Samples 32 96.0
# Reactions 68 204.0
Template (RNA sample) (uL) 68.0 204.0
Sso Fast (uL) 748.0 2,244.0
Pf, 10 uM (uL) 37.4 112.2
Pr, 10 uM (uL) 37.4 112.2
DEPC-treated water (uL) 598.4 1,795.2
Total Volume in mastermix (uL) 1,421.2 4,263.6

Round II

Results look good – only the positive control (DNA sample 1A, in green) had a detected melt temperature. I realized later that I accidentally used the wrong qPCR protocol – I used “CFX_2StepAmp60_EVAGreen+Melt.prcl” instead of “CFX_2StepAmp_EVAGreen+Melt.prcl”. The difference is that step 3 is 60C, while in the correct protocol step 3 is 55C. For my purposes, and since results look good, it seems that this error does not warrant a re-do. I’ll check with the lab, though.

image

Round III

Results look a bit weird, different than before. The fluorescence (RFU) goes up at high temperatures – not sure if this indicates an issue. Also, two samples had melt temperatures – 543 and 475 – but only in 1 rep for each.

Snip20190807_49Snip20190807_48Snip20190807_46

Round IV

Results look okay, except for one well again had a weird increase in RFU at high temps (sample 476, rep 2). However, there was no melt temperature measured in the RNA samples.

Snip20190807_52

All edited CFX files (with sample names, colors) are located in the qPCR-DNA-contamination directory in my O.lurida_Stress repo.

from The Shell Game https://ift.tt/33bv85o
via IFTTT

Laura’s Notebook: DNasing RNA, Round I

According to the QuantSeq library prep protocol, I need to ensure no DNA contamination in my RNA samples. Sam advises that all RNAzol-processed samples will definitely have some DNA contamination. So, I am using the Turbo DNase kit to clean my RNA.

July 31st – DNased RNA, qPCR to identify DNA contamination, batch 1 (n=30)

I ran a first batch of reactions (n=30) using the Turbo DNase kit. The kits we had in the -20 freezer were old (from 2014), so I walked over to the BioSciences stock room (J wing) and purchased 2 new kits (50 reactions per; I’ll need a couple more kits). NOTE: each kit is ~$145.

Turbo DNA Protocol

All reactions used 50 uL RNA, as per the manual’s example. Here were my steps:

  • Labeled 2 sets of 0.5 mL microcentrifuge tubes with RNA sample names
  • Transfered 1 uL of DNase into one set of tubes
  • Transfered 5 uL of Turbo DNase Buffer into each tube
  • Transfered 50 uL of RNA into tubes
  • Mixed gently – used low setting on the vortexer.
  • Incubated at 37C – used the thermocycler in FTR 209 – for 20 minutes
  • Removed samples from thermocycler, added 5 uL inactivation reagent solution. Prior to pipetting this inactivation reagent, I vortexted it thoroughly.
  • Incubated at room temperature for 5 minutes. Vortexed each sample briefly twice during this incubation time to keep solution mixed.
  • Centrifuged samples for 90 seconds at 10,000 rcf
  • Carefully transferred supernatant to fresh, labeled tubes.
  • Held DNased RNA on ice, while I learned how to run qPCR

qPCR to assess DNA contamination

I will use the SsoFast enzyme, a DNA polymerase technology that performs a super fast reaction, and the BIORAD CFX Connect qPCR machine.

Protocol:

  1. Created mastermix for PCR reactions for a total of 64 wells. The following table shows volumes needed for 1 pCCR reaction, then volumes needed for a mastermix for 64 reactions:
per reaction 7/31/19
# Samples 1 30
# Reactions 1.0 62
Template (RNA sample) (uL) 1.0 62.0
Sso Fast (uL) 10.0 682.0
Pf, 10 uM (uL) 0.5 34.1
Pr, 10 uM (uL) 0.5 34.1
DEPC-treated water (uL) 8.0 545.6
Total Volume in mastermix (uL) 20.0 1,357.8

Following Sam’s lab notebook entry, I used elongation factor primers to check for DNA contamination:

  • EF1_qPCR_5’ (SRID 309) (Forward primer, Pf, SRID = 310)
  • EF1_qPCR_3’ (SRID 310) (Reverse primer, Pr, SRID = 309)

These primer stocks are stored in a small fridge in FTR 213, and can be found using the primer database. The stock concentrations are 100 micromolar. I need to use a working stock at 10 uM, so I melted the stocks and diluted 15 uL of each stock in 135 uL DEPC-treated ultrapure water (150 uL total volume).

The total volume per reaction is 20 uL. After creating the mastermix, I pipetted 19 uL of mastermix into a qPCR well plate. NOTE: the type of plate is specific – it’s a white plate, and “low profile” – which is specified on the qPCR software. I then pipetted 1 uL of each sample (i.e. template), in duplicate, to the well plate. I loaded samples horizontally (A1, A2, A3 … etc.) for ease of reading data downstream. In addition to including a control sample, which has been processed alongside the other samples since homogenization (sample 571), I included a No Template Control (NTC, 1uL water added instead of a sample), and DNA isolated from Oly larvae back in March 2018 (sample 69a, RNA sample 8a) as a positive control. To seal the plate I used the clear tape-like cover, rather than the clear plastic caps. I did not vortex the well plate prior to the qPCR reaction.

I carried the well plate over to the qPCR room, loaded it onto the CFX Connect, and opened the MAESTRO software on the adjacent computer. I used the Wizard to help configure the run. Here are the steps to execute the run:

Select “User Defined”

Capture01

Select Protocol: “CFX_2StepAmp_EVAGreen+Melt.prcl”

Capture02

Select plate file: “QuickPlate_96 wells_sybr_white.pltd” – this ensures that all wells are measured. We don’t assign sample names prior to running, but can edit the data file after completion.

Capture03

Select “Next”

Capture04

Select “Save” – it will automatically save the file to Owl and filename will include the run date.

Capture05

This is a screenshot immediately upon protocol initiation

Capture06

I downloaded MAESTRO to my computer (Mac version), and edited the plate setup to include sample names, and color coded melt curves by sample type: GREEN is positive control (n=2); RED is NTC (n=1), and PINK is the homogenization/isolation/DNase control (n=1); BLUE are the samples.

image

Data and report are saved on github in the O.lurida_Stress repo.

The melt curve doesn’t look like Sam’s recent run. However, I realize that the primers were not O. lurida, but were C. gigas. I didn’t think to ask whether the primers needed to be O. lurida specific, but I’m guessing yes. I will plan to move forward with the next batch of Turbo DNase-ing, and will figure out which primers are optimal. Interestingly I did see some DNA, and a melt temperature, for the positive controls, but the fluorescence was not as high as Sam’s example. Also interesting is that my homogenization/isolation control (pink) had a weird peak, suggesting some contamination.

image

Here ares ome qPCR notes from Sam’s instructions:

  • Keep RNA on ice while working with them, and store in -80 always.
  • There are 2x SsoFast aliquots in the fridge, and also in the freezer in the “PCR supplies” box in the -20 (both in FTR 209).
  • Mastermixes should be used the same day they are prepared, but can sit on ice for a few hours.
  • qPCR plates can be prepared, then sealed and held in the fridge for a bit. For example, I could prepare one qPCR plate, then while it is running I can prepare another and hold it in the fridge until the machine is ready again.
  • Always use the button to open/close the BioRAD CFX Connect lid – don’t manually close the lid

Quantified DNased RNA

Used Qubit HS RNA to measure RNA concentration in DNased samples. Approximate volume remaining for DNased RNA is 50 uL. I find it odd that some of my samples have more concentrated RNA after the DNasing. I will look in to that.

Date larvae collected Cohort Treatment TISSUE SAMPLE # Homo./RNA TUBE # VOL RNAzol (mL) MASS TISSUE (mg) [RNA] ng/uL Volume for DNase treatment Amount of RNA in Dnase treatment (ug), max is 10 ug Date Turbo Dnase treatment [RNA] after Turbo Dnase treatment
5/24/17 Dabob Bay 10 Ambient 14-A 401 1 100 52.0 50 2.60 7/31/19 93.4
5/31/17 Dabob Bay 10 Ambient 31-A 402 1 10 140.0 50 7.00 7/31/19 114.0
5/26/17 Dabob Bay 10 Low 23-A 411 1 10 57.2 50 2.86 7/31/19 72.6
5/27/17 Dabob Bay 10 Low 27-A 412 1 10 60.8 50 3.04 7/31/19 31.2
6/12/17 Dabob Bay 6 Ambient 59-A 421 1 10 43.0 50 2.15 7/31/19 57.6
6/7/17 Dabob Bay 6 Low 51-A 431b 1 20 61.2 50 3.06 7/31/19 83.0
6/17/17 Dabob Bay 6 Low 72-A 432 1 50 47.6 50 2.38 7/31/19 74.0
5/25/17 Fidalgo Bay 10 Ambient 20-A 441 1 70 46.0 50 2.30 7/31/19 16.2
6/3/17 Fidalgo Bay 10 Ambient 38-A 442b 1 80 56.2 50 2.81 7/31/19 69.8
5/24/17 Fidalgo Bay 10 Low 16-A 451 1 70 68.4 50 3.42 7/31/19 68.4
5/24/17 Fidalgo Bay 10 Low 18-A 452b 1 80 48.4 50 2.42 7/31/19 97.2
5/26/17 Fidalgo Bay 6 Ambient 22-A 461b 1 100 54.0 50 2.70 7/31/19 84.0
5/29/17 Fidalgo Bay 6 Ambient 29-A 462b 1 60 69.8 50 3.49 7/31/19 106.0
5/25/17 Fidalgo Bay 6 Low 19-A 471b 1 100 71.0 50 3.55 7/31/19 108.0
5/26/17 Fidalgo Bay 6 Low 21-A 472b 1 70 64.0 50 3.20 7/31/19 97.0
5/20/17 Oyster Bay C1 10 Ambient 02-A 481 1 40 64.4 50 3.22 7/31/19 89.2
5/20/17 Oyster Bay C1 10 Ambient 04-A 482 1 60 67.2 50 3.36 7/31/19 22.2
5/23/17 Oyster Bay C1 10 Ambient 09-A 484 1 40 66.2 50 3.31 7/31/19 58.4
6/15/17 Oyster Bay C1 10 Ambient 66-A 491 1 20 126.0 50 6.30 7/31/19 58.4
6/14/17 Oyster Bay C1 10 Low 62-A 506 1 80 63.8 50 3.19 7/31/19 29.2
6/5/17 Oyster Bay C1 6 Ambient 45-A 513 30 156.0 50 7.80 7/31/19 142.0
5/21/17 Oyster Bay C1 6 Low 01-A 521 1 70 54.4 50 2.72 7/31/19 66.6
5/22/17 Oyster Bay C1 6 Low 07-A 522 1 20 60.8 50 3.04 7/31/19 32.2
6/15/17 Oyster Bay C1 6 Low 68-A 528 1 30 162.0 50 8.10 7/31/19 87.6
5/24/17 Oyster Bay C2 10 Ambient 17-A 531 1 60 88.2 50 4.41 7/31/19 95.4
5/23/17 Oyster Bay C2 10 Low 12-A 541 1 40 45.6 50 2.28 7/31/19 44.4
5/24/17 Oyster Bay C2 10 Low 13-A 542 1 30 82.0 50 4.10 7/31/19 32.8
6/3/17 Oyster Bay C2 6 Ambient 41-A 552b 1 80 64.8 50 3.24 7/31/19 74.6
5/21/17 Oyster Bay C2 6 Low 05-A 561 1 40 43.4 50 2.17 7/31/19 28.0
NA RNA Control RNA Control 571 1 10 LOW 50 LOW 7/31/19 LOW

from The Shell Game https://ift.tt/2YJcwq3
via IFTTT

Laura’s Notebook: DNasing RNA

According to the QuantSeq library prep protocol, I need to ensure no DNA contamination in my RNA samples. Sam advises that all RNAzol-processed samples will definitely have some DNA contamination. So, I am using the Turbo DNase kit to clean my RNA.

July 31st – DNased RNA, qPCR to identify DNA contamination, batch 1 (n=30)

I ran a first batch of reactions (n=30) using the Turbo DNase kit. The kits we had in the -20 freezer were old (from 2014), so I walked over to the BioSciences stock room (J wing) and purchased 2 new kits (50 reactions per; I’ll need a couple more kits). NOTE: each kit is ~$145.

Turbo DNA Protocol

All reactions used 50 uL RNA, as per the manual’s example. Here were my steps:

  • Labeled 2 sets of 0.5 mL microcentrifuge tubes with RNA sample names
  • Transfered 1 uL of DNase into one set of tubes
  • Transfered 5 uL of Turbo DNase Buffer into each tube
  • Transfered 50 uL of RNA into tubes
  • Mixed gently – used low setting on the vortexer.
  • Incubated at 37C – used the thermocycler in FTR 209 – for 20 minutes
  • Removed samples from thermocycler, added 5 uL inactivation reagent solution. Prior to pipetting this inactivation reagent, I vortexted it thoroughly.
  • Incubated at room temperature for 5 minutes. Vortexed each sample briefly twice during this incubation time to keep solution mixed.
  • Centrifuged samples for 90 seconds at 10,000 rcf
  • Carefully transferred supernatant to fresh, labeled tubes.
  • Held DNased RNA on ice, while I learned how to run qPCR

qPCR to assess DNA contamination

I will use the SsoFast enzyme, a DNA polymerase technology that performs a super fast reaction, and the BIORAD CFX Connect qPCR machine.

Protocol:

  1. Created mastermix for PCR reactions for a total of 64 wells. The following table shows volumes needed for 1 pCCR reaction, then volumes needed for a mastermix for 64 reactions:
per reaction 7/31/19
# Samples 1 30
# Reactions 1.0 62
Template (RNA sample) (uL) 1.0 62.0
Sso Fast (uL) 10.0 682.0
Pf, 10 uM (uL) 0.5 34.1
Pr, 10 uM (uL) 0.5 34.1
DEPC-treated water (uL) 8.0 545.6
Total Volume in mastermix (uL) 20.0 1,357.8

Following Sam’s lab notebook entry, I used elongation factor primers to check for DNA contamination:

  • EF1_qPCR_5’ (SRID 309) (Forward primer, Pf, SRID = 310)
  • EF1_qPCR_3’ (SRID 310) (Reverse primer, Pr, SRID = 309)

These primer stocks are stored in a small fridge in FTR 213, and can be found using the primer database. The stock concentrations are 100 micromolar. I need to use a working stock at 10 uM, so I melted the stocks and diluted 15 uL of each stock in 135 uL DEPC-treated ultrapure water (150 uL total volume).

The total volume per reaction is 20 uL. After creating the mastermix, I pipetted 19 uL of mastermix into a qPCR well plate. NOTE: the type of plate is specific – it’s a white plate, and “low profile” – which is specified on the qPCR software. I then pipetted 1 uL of each sample (i.e. template), in duplicate, to the well plate. I loaded samples horizontally (A1, A2, A3 … etc.) for ease of reading data downstream. In addition to including a control sample, which has been processed alongside the other samples since homogenization (sample 571), I included a No Template Control (NTC, 1uL water added instead of a sample), and DNA isolated from Oly larvae back in March 2018 (sample 69a, RNA sample 8a) as a positive control. To seal the plate I used the clear tape-like cover, rather than the clear plastic caps. I did not vortex the well plate prior to the qPCR reaction.

I carried the well plate over to the qPCR room, loaded it onto the CFX Connect, and opened the MAESTRO software on the adjacent computer. I used the Wizard to help configure the run. Here are the steps to execute the run:

Select “User Defined”

Capture01

Select Protocol: “CFX_2StepAmp_EVAGreen+Melt.prcl”

Capture02

Select plate file: “QuickPlate_96 wells_sybr_white.pltd” – this ensures that all wells are measured. We don’t assign sample names prior to running, but can edit the data file after completion.

Capture03

Select “Next”

Capture04

Select “Save” – it will automatically save the file to Owl and filename will include the run date.

Capture05

This is a screenshot immediately upon protocol initiation

Capture06

I downloaded MAESTRO to my computer (Mac version), and edited the plate setup to include sample names, and color coded melt curves by sample type: GREEN is positive control (n=2); RED is NTC (n=1), and PINK is the homogenization/isolation/DNase control (n=1); BLUE are the samples.

image

Data and report are saved on github in the O.lurida_Stress repo.

The melt curve doesn’t look like Sam’s recent run. However, I realize that the primers were not O. lurida, but were C. gigas. I didn’t think to ask whether the primers needed to be O. lurida specific, but I’m guessing yes. I will plan to move forward with the next batch of Turbo DNase-ing, and will figure out which primers are optimal. Interestingly I did see some DNA, and a melt temperature, for the positive controls, but the fluorescence was not as high as Sam’s example. Also interesting is that my homogenization/isolation control (pink) had a weird peak, suggesting some contamination.

image

Here ares ome qPCR notes from Sam’s instructions:

  • Keep RNA on ice while working with them, and store in -80 always.
  • There are 2x SsoFast aliquots in the fridge, and also in the freezer in the “PCR supplies” box in the -20 (both in FTR 209).
  • Mastermixes should be used the same day they are prepared, but can sit on ice for a few hours.
  • qPCR plates can be prepared, then sealed and held in the fridge for a bit. For example, I could prepare one qPCR plate, then while it is running I can prepare another and hold it in the fridge until the machine is ready again.
  • Always use the button to open/close the BioRAD CFX Connect lid – don’t manually close the lid

Quantified DNased RNA

Used Qubit HS RNA to measure RNA concentration in DNased samples. Approximate volume remaining for DNased RNA is 50 uL. I find it odd that some of my samples have more concentrated RNA after the DNasing. I will look in to that.

Date larvae collected Cohort Treatment TISSUE SAMPLE # Homo./RNA TUBE # VOL RNAzol (mL) MASS TISSUE (mg) [RNA] ng/uL Volume for DNase treatment Amount of RNA in Dnase treatment (ug), max is 10 ug Date Turbo Dnase treatment [RNA] after Turbo Dnase treatment
5/24/17 Dabob Bay 10 Ambient 14-A 401 1 100 52.0 50 2.60 7/31/19 93.4
5/31/17 Dabob Bay 10 Ambient 31-A 402 1 10 140.0 50 7.00 7/31/19 114.0
5/26/17 Dabob Bay 10 Low 23-A 411 1 10 57.2 50 2.86 7/31/19 72.6
5/27/17 Dabob Bay 10 Low 27-A 412 1 10 60.8 50 3.04 7/31/19 31.2
6/12/17 Dabob Bay 6 Ambient 59-A 421 1 10 43.0 50 2.15 7/31/19 57.6
6/7/17 Dabob Bay 6 Low 51-A 431b 1 20 61.2 50 3.06 7/31/19 83.0
6/17/17 Dabob Bay 6 Low 72-A 432 1 50 47.6 50 2.38 7/31/19 74.0
5/25/17 Fidalgo Bay 10 Ambient 20-A 441 1 70 46.0 50 2.30 7/31/19 16.2
6/3/17 Fidalgo Bay 10 Ambient 38-A 442b 1 80 56.2 50 2.81 7/31/19 69.8
5/24/17 Fidalgo Bay 10 Low 16-A 451 1 70 68.4 50 3.42 7/31/19 68.4
5/24/17 Fidalgo Bay 10 Low 18-A 452b 1 80 48.4 50 2.42 7/31/19 97.2
5/26/17 Fidalgo Bay 6 Ambient 22-A 461b 1 100 54.0 50 2.70 7/31/19 84.0
5/29/17 Fidalgo Bay 6 Ambient 29-A 462b 1 60 69.8 50 3.49 7/31/19 106.0
5/25/17 Fidalgo Bay 6 Low 19-A 471b 1 100 71.0 50 3.55 7/31/19 108.0
5/26/17 Fidalgo Bay 6 Low 21-A 472b 1 70 64.0 50 3.20 7/31/19 97.0
5/20/17 Oyster Bay C1 10 Ambient 02-A 481 1 40 64.4 50 3.22 7/31/19 89.2
5/20/17 Oyster Bay C1 10 Ambient 04-A 482 1 60 67.2 50 3.36 7/31/19 22.2
5/23/17 Oyster Bay C1 10 Ambient 09-A 484 1 40 66.2 50 3.31 7/31/19 58.4
6/15/17 Oyster Bay C1 10 Ambient 66-A 491 1 20 126.0 50 6.30 7/31/19 58.4
6/14/17 Oyster Bay C1 10 Low 62-A 506 1 80 63.8 50 3.19 7/31/19 29.2
6/5/17 Oyster Bay C1 6 Ambient 45-A 513 30 156.0 50 7.80 7/31/19 142.0
5/21/17 Oyster Bay C1 6 Low 01-A 521 1 70 54.4 50 2.72 7/31/19 66.6
5/22/17 Oyster Bay C1 6 Low 07-A 522 1 20 60.8 50 3.04 7/31/19 32.2
6/15/17 Oyster Bay C1 6 Low 68-A 528 1 30 162.0 50 8.10 7/31/19 87.6
5/24/17 Oyster Bay C2 10 Ambient 17-A 531 1 60 88.2 50 4.41 7/31/19 95.4
5/23/17 Oyster Bay C2 10 Low 12-A 541 1 40 45.6 50 2.28 7/31/19 44.4
5/24/17 Oyster Bay C2 10 Low 13-A 542 1 30 82.0 50 4.10 7/31/19 32.8
6/3/17 Oyster Bay C2 6 Ambient 41-A 552b 1 80 64.8 50 3.24 7/31/19 74.6
5/21/17 Oyster Bay C2 6 Low 05-A 561 1 40 43.4 50 2.17 7/31/19 28.0
NA RNA Control RNA Control 571 1 10 LOW 50 LOW 7/31/19 LOW

from The Shell Game https://ift.tt/31c2jUP
via IFTTT