Troubleshooting DNA/RNA Extraction of Pentagona and Hystera Sea Stars
DNA/RNA Extractions on 4 Sea Star Tissue Samples Stored in DNA/RNA Shield, 2 Pentagona and 2 Hystera to Nail Down the Right Extraction Protocol
Notes
- Using the Zymo DNA/RNA Miniprep Plus kit
- Using these beads
- Re-read the Zymo protocol and they do recommend incubating homogenized tissue for 30 min (although they do not for “tough to lyse samples” or for samples stored in DNA/RNA Shield)
- Plan is to cut small tissue pieces for all samples, mince them with a razor blade, homogenize for 1 minute, then do pro K digestion in the bead tube for 30 minutes
Sample Preparation
- Samples:
- CPOI-003
- CPBP-008
- CHSY-007
- CHSB-010
- 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 bead tube per sample with 700ul of DNA/RNA shield in each
- 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
- CPOI-003 tissue piece and minced piece
- CPBP-008 tissue piece and minced piece
- CHSY-007 tissue piece and minced piece
- CHSB-010 tissue piece and minced piece
- Vortexed the bead tubes for 1 minute at max speed
- Samples looked more broken up than last time!
- CPOI-003
- CPBP-008
- CHSY-007
- CGSB-010
- To keep the tissue in there during the digestion, I decided to do the digestion in the bead tubes
- Added 70ul of Pro K buffer and 35ul of Proteinase K
- Vortexed and spin down tubes
- Placed in the thermomixer at 55 degrees C, shaking at 300rpm (did not want to smash the beads around again but wanted the liquid moving)
- Digested for 30 minutes
- Tubes after digestion looked somewhat better, some more than others
- CPOI-003
- CPBP-008
- CHSY-007
- CHSB-010
- Removed all the liquid from the bead tubes into new 1.5mL tubes (~550ul)
DNA Extraction
- Added equal volume (550ul) DNA/RNA lysis buffer to each 1.5mL tube
- Flicked and inverted tubes to mix and spun down
- Warmed 10mM Tris HCl pH 8 and nuclease free water in the thermomixer at 70 degrees C
- 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
- Added the remaining liquid to the yellow spin columns
- Centrifuged 16,000rcf for 30 seconds
- Saved the flow through in the 5mL tubes
- 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 (1100ul) 100% ethanol to each 5mL tube
- 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 for the remaining liquid (2 times) 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 30 seconds
- Discarded flow through (Zymo kit waste)
- Created the DNase mix:
- 75ul DNA digestion buffer * 4 = 300ul
- 5ul DNase I * 4 = 20ul
- 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 warmed 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 another 50ul warmed 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 | 186 RFU | - | - |
| Standard 2 | 20912 RFU | - | - |
| CPOI-003 | 30.2 | 29.6 | 29.9 |
| CPBP-008 | 19.7 | 19.4 | 19.55 |
| CHSY-007 | 19.1 | 18.9 | 19 |
| CHSB-010 | 10.5 | 10.4 | 10.45 |
- RNA
| Sample | Reading 1 (ng/ul) | Reading 2 (ng/ul) | Average RNA (ng/ul) |
|---|---|---|---|
| Standard 1 | 399 RFU | - | - |
| Standard 2 | 8607 RFU | - | - |
| CPOI-003 | 20 | 19.6 | 19.8 |
| CPBP-008 | 17.6 | 17.2 | 17.4 |
| CHSY-007 | 14.8 | 14.2 | 14.5 |
| CHSB-010 | 10.6 | 10.2 | 10.4 |
1% Agarose Gel for DNA Quality
- Followed gel protocol
RNA TapeStation
Notes
- Better yield this time but still low, at least there is RNA in all the samples!
- Might try mincing more and incubating longer, not sure what else to do
Written on October 16, 2020