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.
RNA removal
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.
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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