1 Samples

6C14F (single sample ~10 embryos)
7C14F (single sample ~10 embryos)
6C9F, 7C9F = 67C9F (pooled sample. ~20 embryos)

2 Summary

No success with RNA quantity as measured by Nanodrop or Broad Range Qubit Assay.

I think I should increase starting sample size, and look into alternative sample prep for delicate embryos (as compared with adult coral fragments).

I can’t be sure that waiting too long during the DNase Treatment didn’t degrade all the RNA, next round be careful not to go over the 15 minute DNase I incubation time.

3 Extraction Notes

date: 12-AUG-2024

kit: Zymo Quick-DNA/RNA Miniprep Plus Kit

Followed protocol according to my post [M. Capitata Embryo DNA/RNA Extractions Protocol] (https://sarahtanja.github.io/quarto-blog/posts/projects/coral-embryo-leachate/embryo-extractions.html#proteinase-k-digestion) with these changes:

Lysed samples via Bead Bashing in a full 2mL Zymo BeadBashing (0.1 - 0.5mm glass bead) Tube with a total volume of 1mL of DNA/RNA Shield
Transfered 500uL of cleared supernatent to a new nuclease-free tube, making sure not to disturb beads and debris at the bottom of the bead-bashing tube
Conducted Proteinase K digestion as follows:
- Get the Proteinase K out of the -20 freezer & set the heat-block to warm up to 55C for 30mins
- After bead bashing, tubes are ‘intensely bubbly’, to tamp down bubbles, centrifuge in the mini-centrifuge for 1min
- Transfer 500uL of supernatent to a new nuclease-free tube, making sure not to disturb beads and debris at the bottom of the bead-bashing tube2
- Add Proteinase K & Buffer
- Add the appropriate volume of Pro K buffer and Proteinase K (Proteinase K is stored in the -20 after being reconstituted)
- (10:1 ratio of sample:digestion buffer) & (2:1 ratio of digestion buffer:Proteinase K)
- For tubes with approximately 500uL of sample add:
- 50ul pro K buffer 25ul proteinase K Vortex to mix
- Incubate in the heat-block for 30mins at 55C
- Vortex to mix & centrifuge on max for 2mins to pellet any debris
- Transfer 350uL of the cleared supernatent to a new 1.5mL nuclease-free tube, making sure not to disturb any debris at the bottom of the tube
- Add 350uL of DNA/RNA Lysis Buffer to the supernatent(1:1) and vortex to mix
I let DNase I treatment incubate at room temperature for 1 hour (instead of 15 minutes)

eluted DNA volume: 30uL in Zymo DNase-RNase Free Water

eluted RNA volume: 30uL in Zymo DNase-RNase Free Water

4 Nanodrop

4.1 RNA

sample_id	ng/uL	A260	A280	260/280	260/230
6C14F	1.6	0.039	0.008	5.12	5.40
7C14F	3.2	0.081	0.044	1.82	0.86
67C9F	1.0	0.025	0.002	10.94	1.12

5 Qubit

5.1 RNA

Using the Invitrogen Thermo Fischer RNA Broad Range Assay Kit.

Prepared working solution for 3 samples and two standards:

Qubit RNA BR reagent in DMSO = 1uL * 5 = 5uL

Qubit RNA BR Buffer = 199uL * 5 = 995uL

standard 1:

standard 2:

RunID: 2024-08-13_062825

sample_id	qubit_rna_1	qubit_rna_2
6C14F	too low	too low
7C14F	too low	too low
67C9F	too low	too low

6 Storage Location

RNA samples are stored in -80C ‘Old Friedman’ Sanyo freezer on shelf 1 in coral-embryo-leachate RNA wax freezer boxes with green label tape.

DNA samples (not quantified) are stored in -20C in JPG Lab FSH 236 in shelf 2 in coral-embryo-leachate DNA wax freezer box with yellow label tape.

Protein flow-through (not quantified) are stored in -20C in JPG Lab FSH 236 in shelf 2 in coral-embryo-leachate PROTEIN wax freezer box with blue label tape.

--- title: "12-Aug-2024 Embryo RNA Extractions" author: "Sarah Tanja" date: 2024-08-12 date-format: long date-modified: today categories: [lab records] draft: false image: "frozen-vials.jpg" --- # Samples - 6C14F (single sample \~10 embryos) - 7C14F (single sample \~10 embryos) - 6C9F, 7C9F = 67C9F (pooled sample. \~20 embryos) # Summary No success with RNA quantity as measured by Nanodrop or Broad Range Qubit Assay. I think I should increase starting sample size, and look into alternative sample prep for delicate embryos (as compared with adult coral fragments). I can't be sure that waiting too long during the DNase Treatment didn't degrade all the RNA, next round be careful not to go over the 15 minute DNase I incubation time. # Extraction Notes date: 12-AUG-2024 kit: Zymo Quick-DNA/RNA Miniprep Plus Kit Followed protocol according to my post \[M. Capitata Embryo DNA/RNA Extractions Protocol\] (https://sarahtanja.github.io/quarto-blog/posts/projects/coral-embryo-leachate/embryo-extractions.html#proteinase-k-digestion) with these changes: - Lysed samples via Bead Bashing in a full 2mL Zymo BeadBashing (0.1 - 0.5mm glass bead) Tube with a total volume of 1mL of DNA/RNA Shield - Transfered 500uL of cleared supernatent to a new nuclease-free tube, making sure not to disturb beads and debris at the bottom of the bead-bashing tube - Conducted Proteinase K digestion as follows: - Get the Proteinase K out of the -20 freezer & set the heat-block to warm up to 55C for 30mins - After bead bashing, tubes are ‘intensely bubbly’, to tamp down bubbles, centrifuge in the mini-centrifuge for 1min - Transfer 500uL of supernatent to a new nuclease-free tube, making sure not to disturb beads and debris at the bottom of the bead-bashing tube2 - Add Proteinase K & Buffer - Add the appropriate volume of Pro K buffer and Proteinase K (Proteinase K is stored in the -20 after being reconstituted) - (10:1 ratio of sample:digestion buffer) & (2:1 ratio of digestion buffer:Proteinase K) - For tubes with approximately 500uL of sample add: - 50ul pro K buffer 25ul proteinase K Vortex to mix - Incubate in the heat-block for 30mins at 55C - Vortex to mix & centrifuge on max for 2mins to pellet any debris - Transfer 350uL of the cleared supernatent to a new 1.5mL nuclease-free tube, making sure not to disturb any debris at the bottom of the tube - Add 350uL of DNA/RNA Lysis Buffer to the supernatent(1:1) and vortex to mix - I let DNase I treatment incubate at room temperature for 1 hour (instead of 15 minutes) eluted DNA volume: 30uL in `Zymo DNase-RNase Free Water` eluted RNA volume: 30uL in `Zymo DNase-RNase Free Water` # Nanodrop ## RNA | sample_id | ng/uL | A260 | A280 | 260/280 | 260/230 | |-----------|-------|-------|-------|---------|---------| | 6C14F | 1.6 | 0.039 | 0.008 | 5.12 | 5.40 | | 7C14F | 3.2 | 0.081 | 0.044 | 1.82 | 0.86 | | 67C9F | 1.0 | 0.025 | 0.002 | 10.94 | 1.12 | ![](nanodrop_12AUG2024.png) # Qubit ## RNA Using the Invitrogen Thermo Fischer RNA Broad Range Assay Kit. Prepared working solution for 3 samples and two standards: Qubit RNA BR reagent in DMSO = 1uL \* 5 = 5uL Qubit RNA BR Buffer = 199uL \* 5 = 995uL standard 1: standard 2: RunID: 2024-08-13_062825 | sample_id | qubit_rna_1 | qubit_rna_2 | |-----------|-------------|-------------| | 6C14F | too low | too low | | 7C14F | too low | too low | | 67C9F | too low | too low | # Storage Location RNA samples are stored in -80C 'Old Friedman' Sanyo freezer on shelf 1 in `coral-embryo-leachate RNA` wax freezer boxes with green label tape. DNA samples (not quantified) are stored in -20C in JPG Lab FSH 236 in shelf 2 in `coral-embryo-leachate DNA` wax freezer box with yellow label tape. Protein flow-through (not quantified) are stored in -20C in JPG Lab FSH 236 in shelf 2 in `coral-embryo-leachate PROTEIN` wax freezer box with blue label tape.