Laura’s Notebook: QuantSeq Library Prep test-run

Katherine suggested I work through the library prep protocol with a few samples to practice and work out kinks. From her experience, the libraries she generated later in the game were of higher quaity. I’m generating 8 test libraries – this was her recommendation based on the heavy use of 8-channel pipettes.

Following the QuantSeq manual and tips from Katherine.

Quick reference for Libary Generation

image

Notes from this test run:

Started on 8/20/2019

Prior to beginning, I created programs on the PTC-200 DNA Engine Cycler. Programs are labeled “Quantseq-#”, with the # corresponding to step number in the QuantSeq manual.

first strand cDNA synthesis

  • Based on Katherine’s suggestions, I will generate 40 libraries at one, in 5 rows of 8 on a PCR plate. When adding solutions to each well, rather than using a single channel pipette and transferring solutions to individual wells, I’m going to use PCR strips, load enough of each solution into 8 wells (with a bit excess), and use a multichannel to distribute. This way I can hopefuly reduce time.
  • Between each step I need to quickly “spin down” my PCR plate. Our benchtop centrifuge has a minimum run time of 1 minute, and with the time it takes to accelerate and decelerate total time is ~4 minutes. The “quickly spin down” instructions do not define a centrifuge speed to use, so I set it to 3 rcf based on whatt is specified in the equipmentt list – “Benchtop centrifuge (3,000 x g, rotor compatible with 96-well plates”. To reduce the amount of time to spin down plates, I should either set the speed lower, OR simply start, then stop the centrifuge manually.

RNA removal

  • No notes.

second strand cDNA synthesis

  • SS1 and USS are indeed viscous; hold pipette in place when pulling volumes.
  • SAFE STOPPING POINT

Placed in -20C overnight.

Purificaion

(this is 1 of 2 purifications, dubbed “pre-PCR”)

  • As Katherine hinted, it’s important to have a magnetic plate that fits the PCR plates used. The plates we have just have rods – no wells for plates to hold plates in place, which doesn’t work. I ordered a magnet/plate from ebay to hopefully improve the process.
  • SAFE STOPPING POINT.

Placed in -20 on 8/21/19 until next step (qPCR assay).

qPCR assay for optimal # cycles

Performed on 9/3/2019

  • Created a custom qPCR protocol with Sam’s help –
qPCR Assay Mastermis Calcs
Item per rxn (uL) all rxns (inc. NTC) (uL) all rxns * 1.1
Number of samples 1 9
cDNA, diluted to 19uL 1.7 15.3 16.8
PCR mix (PCR) 7 63 69.3
P7 Primer (7000) 5 45 49.5
Enzyme mix (E) 1 9 9.9
2.5x SYBR Green I nucleic acid dye 1.2 10.8 11.9
Elution Buffer (EB) 14.1 126.9 139.6
Mastermix total vol 28.3 254.7 280.2
SYBR Green Calcs Volumes
Stock concentration 100
Desired concentratino 2.5
Total volume needed 2.5x 11.9
Volume 100x 0.2970
Volume DMSO 11.58
Dilution ratio (should be 1:40) 0.025
Final concentration 2.5

Results: Located on Owl, https://ift.tt/2A2cccd with today’s date.

No amplification :/ max RFU should be ~10×10^12, and my no-template control (NTC) has same curve as samples.

2019-09-03_ qPCR-assay-test-run

First troubleshooting step is to see if I actually synthesized cDNA – I believe I can use the Qubit for that. If yes, then I messed up the qPCR somehow (wouldn’t surprise me). Perhaps there is an issue with bubbles? Maybe BioRad settings needed be adjusted for sybr green?

I ordered a trial QuantSeq kit (n=4) for trouble shooting, and as a result spoke with the WA respresentative. Notes from our call:

  • If cDNA synthesis did not occur, then I most likely had contamination with organics or salts, which can inhibit 1st strand synthesis and cause cDNA to degrade when trying to degrade RNA.
  • The TurboDNase method might be an issue, since it did not include a column.
  • Using a cleaner column on existing RNA may do the trick. I did order one box of Zymo Cleaner-Concentrator (n=50), so could run all my samples through this column.
  • 500 ng of input RNA is not necesary (she said that “no one uses that much”). 100 ng should be adequate!

I will receive the trial kit tomorrow, and Kristy is connecting me with the tech support guy.

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