2nd RNA Extraction of Pentagona and Hystera Sea Stars, 7 Samples
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 003
- CPAB2 008
- CPDB 002
- CPDB 009
- CPAB 005
- CPBO 002
- CPBO 006
- 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 003:
- After homogenization and incubation CPAB2 008:
- After homogenization and incubation CPDB 002:
- After homogenization and incubation CPDB 009:
- After homogenization and incubation CPAB 005:
- After homogenization and incubation CPBO 002:
- After homogenization and incubation CPBO 006:
- 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
- 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
- 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 * 7 = 525ul
- 5ul DNase I * 7 = 35ul
- 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 50ul 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 30ul 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
| Sample |
Reading 1 (ng/ul) |
Reading 2 (ng/ul) |
Average DNA (ng/ul) |
| Standard 1 |
187 RFU |
- |
- |
| Standard 2 |
21525 RFU |
- |
- |
| CPAB2 003 |
32.6 |
32.4 |
32.5 |
| CPAB2 008 |
39.8 |
39.8 |
39.8 |
| CPDB 002 |
61.8 |
61.4 |
61.6 |
| CPDB 009 |
48.8 |
48.6 |
48.7 |
| CPAB 005 |
70 |
68.8 |
69.4 |
| CPBO 002 |
25 |
24.8 |
24.9 |
| CPBO 006 |
51.4 |
51.2 |
51.3 |
| Sample |
Reading 1 (ng/ul) |
Reading 2 (ng/ul) |
Average RNA (ng/ul) |
| Standard 1 |
390 RFU |
- |
- |
| Standard 2 |
8007 RFU |
- |
- |
| CPAB2 003 |
10 |
10 |
10 |
| CPAB2 008 |
too low |
- |
- |
| CPDB 002 |
16.6 |
16.6 |
16.6 |
| CPDB 009 |
12.4 |
12.4 |
13.4 |
| CPAB 005 |
20 |
20.2 |
20.1 |
| CPBO 002 |
10.8 |
10.6 |
10.7 |
| CPBO 006 |
14.6 |
14.4 |
14.5 |
RNA TapeStation
- Followed RNA tapestation protocol
- TapeStation Report link
- I decided not to tapestation CPAB 008 even though there was probably some RNA in there, I didn’t want to waste the space on the screentape. I will have to extract one more sample from CPAB2 and then I will have finished with the RNA
Written on November 24, 2020
