3rd RNA Extraction of Pentagona and Hystera Sea Stars, 2 Samples
RNA Extractions of 2 Pentagona With the Zymo DNA/RNA Miniprep Plus kit, doing 2 CPAB2 Samples Because They Have Been So Tough to Get Enough RNA
Notes
- Using the Zymo DNA/RNA Miniprep Plus kit
- Used 1.4mm ceramic beads and the tissuelyser for 2 minutes at 25Hz, stopped at 30 seconds and moved the tubes to the other side of the sample holder in the lyser.
- Incubated at room temp with the proteinase K for 15 minutes shaking at 550rpm
Sample Preparation
- Samples:
- CPAB2 010
- CPAB2 005
- Thawed samples on ice bucket
- Prepared forceps, foil, and razor blades
- Cleaned forceps with 10% bleach, DI water, 70% ethanol, and RNaseZap before and between each sample
- Made 1 ceramic bead tube with 700ul DNA/RNA shield for each tissuelyser sample
- Cut a small piece of tissue for each sample then minced with the razor blade until it was flat and could come apart a little bit and placed in their respective bead homogenization tube
- Ran the tissuelyser for 2 minutes at 25Hz
- Spun down the bead tubes in the minifuge to remove bubbles
- Added 70ul proteinase K digestion buffer and 35ul proteinase K to each bead tube
- Briefly vortexed and spun down sample tubes
- Placed in the thermomixer for 15 minutes shaking at 550rpm at 23 degrees C temp (room temp)
- After homogenization and incubation CPAB2 010:
- After homogenization and incubation CPAB2 005:
- Removed all the supernatant (~725ul) into new 1.5mL tubes
- Centrifuged tubes for 1 minute at 16,00rcf
- Removed liquid (~725ul) into new 1.5mL tubes avoiding small pellet
- Added equal volume (725ul) DNA/RNA lysis buffer to the 5mL tubes
- Vortexed and spun down tubes
DNA Extraction
- Flicked and inverted tubes to mix and spun down
- Warmed 10mM Tris HCl pH 8 in the thermomixer at 70 degrees C note did not warm H2O this time
- Added 700ul of the liquid to yellow spin columns and collection tubes
- Centrifuged 16,000rcf for 30 seconds
- Saved the flow through in new 5mL tubes
- Repeated addition, centrifugation, and saving of flowthrough for the remaining amount of liquid (3 total spins)
- Added 400ul DNA/RNA prep buffer to the yellow spin columns
- Centrifuged 16,000rcf for 30 seconds
- Discarded flow through (Zymo kit waste)
- Added 700ul DNA/RNA wash buffer to the yellow spin columns
- Centrifuged 16,000rcf for 30 seconds
- Discarded flow through (Zymo kit waste)
- Added 400ul DNA/RNA wash buffer to the yellow spin columns
- Centrifuged 16,000rcf for 2 minutes
- Transferred spin columns to new 1.5mL tubes for each sample labeled as final DNA tubes
- Discarded flow through and collection tubes
- Added 50ul warmed 10mM Tris HCl to the spin columns gently by dripping over the filter
- Incubated at room temp for 5 minutes
- Centrifuged 16,000rcf for 30 seconds
- Added another 50ul warmed 10mM Tris HCl to the spin columns gently by dripping over the filter
- Incubated at room temp for 5 minutes
- Centrifuged 16,000rcf for 30 seconds
- Discarded the spin columns
- Made strip tubes with 7ul of each sample in them for QC
- Kept tubes on ice bucket then stored in -20 degree freezer
RNA Extraction
- Added equal volume (1450ul) 100% ethanol to the 5mL tubes of flowthrough
- Vortexed and spun down
- Added 700ul of the liquid to green spin columns and collection tubes
- Centrifuged 16,000rcf for 30 seconds
- Discarded flow through (Zymo kit waste)
- Repeated addition, centrifugation, and saving of flowthrough for the remaining amount of liquid (5 total spins)
- Added 400ul DNA/RNA wash buffer to the green spin columns
- Centrifuged 16,000rcf for 30 seconds
- Discarded flow through (Zymo kit waste)
- Created the DNase mix:
- 75ul DNA digestion buffer * 2 = 150ul
- 5ul DNase I * 2 = 10ul
- Flicked and spun down mix
- Added 80ul DNAse mix to each green spin column filter
- Incubated for 15 minutes at room temp
- Added 400ul DNA/RNA prep buffer to the green spin columns
- Centrifuged 16,000rcf for 30 seconds
- Discarded flow through (Zymo kit waste)
- Added 700ul DNA/RNA wash buffer to the green spin columns
- Centrifuged 16,000rcf for 30 seconds
- Discarded flow through (Zymo kit waste)
- Added 400ul DNA/RNA wash buffer to the green spin columns
- Centrifuged 16,000rcf for 2 minutes
- Transferred spin columns to new 1.5mL tubes for each sample labeled as final RNA tubes
- Discarded flow through and collection tubes
- Added 40ul room temp nuclease free water to the spin columns gently by dripping over the filter
- Incubated at room temp for 5 minutes
- Centrifuged 16,000rcf for 30 seconds
- Added 40ul room temp nuclease free water to the spin columns gently by dripping over the filter
- Incubated at room temp for 5 minutes
- Centrifuged 16,000rcf for 30 seconds
- Discarded the spin columns
- Made strip tubes with 5ul of each sample in them for QC
- Kept tubes on ice bucket then stored in -80 degree freezer
QC
Broad Range Qubit for DNA and RNA
- Followed Qubit protocol
- DNA
| Sample | Reading 1 (ng/ul) | Reading 2 (ng/ul) | Average DNA (ng/ul) |
|---|---|---|---|
| Standard 1 | 183 RFU | - | - |
| Standard 2 | 20062 RFU | - | - |
| CPAB2 010 | 25.4 | 25.2 | 25.3 |
| CPAB2 005 | 15.3 | 15.1 | 15.2 |
- RNA
| Sample | Reading 1 (ng/ul) | Reading 2 (ng/ul) | Average RNA (ng/ul) |
|---|---|---|---|
| Standard 1 | 399 RFU | - | - |
| Standard 2 | 8555 RFU | - | - |
| CPAB2 010 | too low | - | - |
| CPAB2 005 | too low | - | - |
Decided to combine this CPAB2 005 RA extraction and the CPAB2 RNA extraction from 20201118 and vacufuge for 40 minute to concentrate and then re-quantify and do the tapestation
Second Qubit
| Sample | Reading 1 (ng/ul) | Reading 2 (ng/ul) | Average RNA (ng/ul) |
|---|---|---|---|
| Standard 1 | 396 RFU | - | - |
| Standard 2 | 8378 RFU | - | - |
| CPAB2 005 | 13.2 | 13 | 13.1 |
Total volume is ~63ul
RNA TapeStation
- Followed RNA tapestation protocol
- Kevin ran my 1 sample along with some he had to do: mine is the last sample
- TapeStation Report link
DNA Gel
Written on December 1, 2020