1 Samples

1H9F, 2H9F, 3H9F = 123H9
4M4F, 5M4F, 6M4F = 456M4
7L4F, 8L4F, 9L4F = 789L4
7M14F, 8M14F, 9M14F = 789M14
10C4F, 11C4F, 12C4F = 101112C4
10H14F, 11H14F, 12H14F = 101112H14
13C9F, 14C9F, 15C9F = 131415C9
13C14F, 14C14F, 15C14F = 131415C14

2 Summary

Note

Overall success! Big change here was homogenizing for 10 minutes, the average yield did seem to improve and I got clear supernatent after the homogenization step. Working with 8 samples at a time was manageable. Still seeing quantity on the lower workable end, but I think that’s an artifact of working with small embryo sample starting material size.

3 Extraction Notes

kit: Zymo Quick-DNA/RNA Miniprep Plus Kit

Thaw samples to room temp
Transfer and pool samples to one Zymo BashingBead Tube
Homogenize for 10 minutes
- 10 min Zymo BashingBead Tube
  - The 4hpf samples are hardier and take a little longer to lyse than the other embryonic phases
Centrifuge at 16,000xg for 1 min to get rid of bubbles
- Check for complete lysis of embryos
- Supernatent is clear!
Transfer 2 550uL aliquots of cleared supernatent to one new nuclease-free tube
Centrifuge at 16,000xg for 1 min to pellet any beads
Split cleared supernatent into two aliquots of 530uL & transfer each aliquot to a new nuclease-free tube
Add 530uL of DNA/RNA Lysis Buffer to each tube ( a total of 1060uL volume in each tube )
Vortex to mix for 5 seconds

—> Proceed to purification
Sequentially transfer 1060uL of each sample in aliquots of 700uL into yellow Spin-Away Filter in a Collection Tube and centrifuge for 30s at 16,000xg SAVE THE FLOW THROUGH FOR RNA PURIFICATION by tipping flow through into spare collection tube (15mL Falcon Tube)
Doubled volume of 100% (200 proof) ethanol to the RNA flow through in a falcon tube and vortexed to mix. End volume of flow through + ethanol = 4mL
Sequentially transfer RNA flow-through + ethanol in volumes =>700uL into the green Zymo Spin IIICG Column in a Collection Tube. This should take 6 ‘spins’
Saved 1st 2 spins (1400uL) for protein tube. Once collected place in -20 freezer.

Performed DNase I Treatment on green RNA spin column

DB = 75*8
print(DB)

[1] 600

Dnase = 5*8 
print(Dnase)

[1] 40

Note

    I didn't invert to mix... I always lose some volume 'beads' in the lid when I do this. Instead I flicked the tube to mix it and kept all the volume down in the conical end of the tube. This worked well and I had a full 80uL of treatment per spin column to work with!

400uL of Prep Buffer
700uL of DNA/RNA Wash Buffer
400uL of DNA/RNA Wash Buffer, 2min
Centrifuge 2min dry run
Warmed Zymo DNase-RNase Free Water to 55C in heat block before elution, and slowly dripped 50uL of water directly over filter.

4 End Products

eluted DNA volume: 50uL in Zymo DNase-RNase Free Water

eluted RNA volume: 50uL in warmed (55C) Zymo DNase-RNase Free Water

Important

I upped the elution volume from 30 to 50 for RNA because I wanted to make sure the nuclease-free water volume was enough to saturate the whole filter and get the RNA from it… The minimum volume in the kit instructions is 50uL… And I do have an RNA Clean & Concentrator kit that allows me to reduce that if needed. As long as total RNA is at least 100ng, we’re good. That means that qubit value (ng/uL) times the elution volume.

5 Qubit QAQC

Using the Invitrogen Thermo Fischer RNA High Sensitivity Assay Kit.

Prepared working solution for 6 samples and two standards (plus some extra) in a 5mL tube:

buffer <- 199*10
print(buffer)

[1] 1990

reagent <- 1*10
print(reagent)

[1] 10

5.0.1 RNA

standard 1: 92.70

standard 2: 1770.25

RunID: 2024-08-21_054905

sample_id	qubit_rna_1	qubit_rna_2
123H9	6.66 ng/uL	6.68 ng/uL
456M4	9.60 ng/uL	9.52 ng/uL
789L4	8.18 ng/uL	8.18 ng/uL
789M14	10.6 ng/uL	10.4 ng/uL
101112C4	15.4 ng/uL	15.4 ng/uL
101112H14	7.20 ng/uL	7.02 ng/uL
131415C9	10.7 ng/uL	10.8 ng/uL
131415C14	11.4 ng/uL	11.4 ng/uL

6 Storage Location

RNA samples are stored in -80C ‘JPG Lab Thermo’ 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: "20-Aug-2024 Embryo RNA Extractions" author: "Sarah Tanja" date: 08/20/2024 date-format: long date-modified: today categories: [lab records] draft: false toc: true toc-exand: true toc-title: Contents <i class="bi bi-bookmark-heart"></i> toc-depth: 5 toc-location: left reference-location: margin citation-location: margin link-external-icon: true link-external-newwindow: true --- # Samples - 1H9F, 2H9F, 3H9F = 123H9 - 4M4F, 5M4F, 6M4F = 456M4 - 7L4F, 8L4F, 9L4F = 789L4 - 7M14F, 8M14F, 9M14F = 789M14 - 10C4F, 11C4F, 12C4F = 101112C4 - 10H14F, 11H14F, 12H14F = 101112H14 - 13C9F, 14C9F, 15C9F = 131415C9 - 13C14F, 14C14F, 15C14F = 131415C14 # Summary ::: callout-note Overall success! Big change here was homogenizing for 10 minutes, the average yield did seem to improve and I got clear supernatent after the homogenization step. Working with 8 samples at a time was manageable. Still seeing quantity on the lower workable end, but I think that's an artifact of working with small embryo sample starting material size. ::: # Extraction Notes kit: Zymo Quick-DNA/RNA Miniprep Plus Kit - Thaw samples to room temp - Transfer and pool samples to one Zymo BashingBead Tube - Homogenize for 10 minutes - 10 min Zymo BashingBead Tube - The 4hpf samples are hardier and take a little longer to lyse than the other embryonic phases - Centrifuge at 16,000xg for 1 min to get rid of bubbles - Check for complete lysis of embryos - Supernatent is clear! - Transfer 2 550uL aliquots of cleared supernatent to one new nuclease-free tube - Centrifuge at 16,000xg for 1 min to pellet any beads - Split cleared supernatent into two aliquots of 530uL & transfer each aliquot to a new nuclease-free tube - Add 530uL of DNA/RNA Lysis Buffer to each tube ( a total of 1060uL volume in each tube ) - Vortex to mix for 5 seconds ---\> Proceed to purification - Sequentially transfer 1060uL of each sample in aliquots of 700uL into yellow Spin-Away Filter in a Collection Tube and centrifuge for 30s at 16,000xg **SAVE THE FLOW THROUGH FOR RNA PURIFICATION by tipping flow through into spare collection tube (15mL Falcon Tube)** - Doubled volume of 100% (200 proof) ethanol to the RNA flow through in a falcon tube and vortexed to mix. End volume of flow through + ethanol = 4mL - Sequentially transfer RNA flow-through + ethanol in volumes =\>700uL into the green Zymo Spin IIICG Column in a Collection Tube. This should take 6 'spins' - Saved 1st 2 spins (1400uL) for protein tube. Once collected place in -20 freezer. - Performed DNase I Treatment on green RNA spin column ```{r} DB = 75*8 print(DB) Dnase = 5*8 print(Dnase) ``` ::: callout-note ``` I didn't invert to mix... I always lose some volume 'beads' in the lid when I do this. Instead I flicked the tube to mix it and kept all the volume down in the conical end of the tube. This worked well and I had a full 80uL of treatment per spin column to work with! ``` ::: - 400uL of Prep Buffer - 700uL of DNA/RNA Wash Buffer - 400uL of DNA/RNA Wash Buffer, 2min - Centrifuge 2min dry run - Warmed Zymo DNase-RNase Free Water to 55C in heat block before elution, and slowly dripped 50uL of water directly over filter. # End Products eluted DNA volume: 50uL in `Zymo DNase-RNase Free Water` eluted RNA volume: 50uL in warmed (55C) `Zymo DNase-RNase Free Water` ::: callout-important I upped the elution volume from 30 to 50 for RNA because I wanted to make sure the nuclease-free water volume was enough to saturate the whole filter and get the RNA from it... The minimum volume in the kit instructions is 50uL... And I do have an RNA Clean & Concentrator kit that allows me to reduce that if needed. As long as total RNA is at least 100ng, we're good. That means that qubit value (ng/uL) times the elution volume. ::: # Qubit QAQC Using the Invitrogen Thermo Fischer RNA High Sensitivity Assay Kit. Prepared working solution for 6 samples and two standards (plus some extra) in a 5mL tube: ```{r} buffer <- 199*10 print(buffer) reagent <- 1*10 print(reagent) ``` ### RNA standard 1: 92.70 standard 2: 1770.25 RunID: 2024-08-21_054905 +---------------+-------------+-------------+ | sample_id | qubit_rna_1 | qubit_rna_2 | +===============+=============+=============+ | - 123H9 | 6.66 ng/uL | 6.68 ng/uL | +---------------+-------------+-------------+ | - 456M4 | 9.60 ng/uL | 9.52 ng/uL | +---------------+-------------+-------------+ | - 789L4 | 8.18 ng/uL | 8.18 ng/uL | +---------------+-------------+-------------+ | - 789M14 | 10.6 ng/uL | 10.4 ng/uL | +---------------+-------------+-------------+ | - 101112C4 | 15.4 ng/uL | 15.4 ng/uL | +---------------+-------------+-------------+ | - 101112H14 | 7.20 ng/uL | 7.02 ng/uL | +---------------+-------------+-------------+ | - 131415C9 | 10.7 ng/uL | 10.8 ng/uL | +---------------+-------------+-------------+ | - 131415C14 | 11.4 ng/uL | 11.4 ng/uL | +---------------+-------------+-------------+ # Storage Location RNA samples are stored in -80C 'JPG Lab Thermo' 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: