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
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.
- 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.
(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|
|Total volume needed 2.5x||11.9|
|Dilution ratio (should be 1:40)||0.025|
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.
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.