1 Samples

1L4F, 2L4F, 3L4F = 123L4
- 1 min Zymo BashingBead Tube
7L9F, 8L9F, 9L9F = 789L9
- 1 min Zymo BashingBead Tube
10C9F, 11C9F, 12C9F = 101112C9
- 1 min Zymo BashingBead Tube
10H9F, 11H9F, 12H9F = 101112H9
- 1 min Zymo BashingBead Tube
13M14F, 14M14F, 15M14F = 131415M14
- 1 min Zymo BashingBead Tube
13C4F, 14C4F, 15C4F = 131415C4
- 1 min Zymo BashingBead Tube

2 Summary

Note

3 Extraction Notes

date: 16-AUG-2024

kit: Zymo Quick-DNA/RNA Miniprep Plus Kit

Thaw samples to room temp
Vortex to mix
Transfer to Zymo BashingBead Tube
Homogenize for 3 minutes
- The 4hpf samples are hardier and take a little longer to lyse than the other embryonic phases
Centrifuge at 16,000xg for 30 seconds to get rid of bubbles
- Check for complete lysis of embryos
Transfer 2 550uL aliquots of cleared supernatent to a new nuclease-free tube
Centrifuge at 16,000xg for 1 min to pellet any beads
Split cleared supernatent into two aliquots of 500uL & transfer each aliquot to a new nuclease-free tube
Added 500uL of DNA/RNA Lysis Buffer to each tube ( a total of 1000uL volume in each tube )
Vortex to mix for 5 seconds

—> Proceed to purification
Sequentially transfer 1000uL 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

Caution

Forgot to run DNA/RNA Wash prior to DNase I treatment!!!
```
DB = 75*6
print(DB)
```
```
[1] 450
```
```
Dnase = 5*6 
print(Dnase)
```
```
[1] 30
```
- 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 cnical end of the tube. This worked well and I had a full 80uL of treatment per spin column to work with.
Warmed Zymo DNase-RNase Free Water to 55C in heat block before elution, and slowly dripped water directly over filter.

eluted DNA volume: 30uL 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.

4 Qubit

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

4.0.1 RNA

standard 1: 96.43

standard 2: Error… re-run cal standards? No…

RunID: 2024-08-17_045016

sample_id	qubit_rna_1	qubit_rna_2
123L4	10.9 ng/uL	10.9 ng/uL
789L9	6.32 ng/uL	6.26 ng/uL
101112C9	5.56 ng/uL	5.46 ng/uL
101112H9	8.46 ng/uL	8.46 ng/uL
131415M14	7.38 ng/uL	7.32 ng/uL
131415C4	11.1 ng/uL	11.1 ng/uL

5 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: "16-Aug-2024 Embryo RNA Extractions" author: "Sarah Tanja" date: 08/16/2024 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 - 1L4F, 2L4F, 3L4F = 123L4 - 1 min Zymo BashingBead Tube - 7L9F, 8L9F, 9L9F = 789L9 - 1 min Zymo BashingBead Tube - 10C9F, 11C9F, 12C9F = 101112C9 - 1 min Zymo BashingBead Tube - 10H9F, 11H9F, 12H9F = 101112H9 - 1 min Zymo BashingBead Tube - 13M14F, 14M14F, 15M14F = 131415M14 - 1 min Zymo BashingBead Tube - 13C4F, 14C4F, 15C4F = 131415C4 - 1 min Zymo BashingBead Tube # Summary ::: callout-note ::: # Extraction Notes date: 16-AUG-2024 kit: Zymo Quick-DNA/RNA Miniprep Plus Kit - Thaw samples to room temp - Vortex to mix - Transfer to Zymo BashingBead Tube - Homogenize for 3 minutes - The 4hpf samples are hardier and take a little longer to lyse than the other embryonic phases - Centrifuge at 16,000xg for 30 seconds to get rid of bubbles - Check for complete lysis of embryos - Transfer 2 550uL aliquots of cleared supernatent to a new nuclease-free tube - Centrifuge at 16,000xg for 1 min to pellet any beads - Split cleared supernatent into two aliquots of 500uL & transfer each aliquot to a new nuclease-free tube - Added 500uL of DNA/RNA Lysis Buffer to each tube ( a total of 1000uL volume in each tube ) - Vortex to mix for 5 seconds ---\> Proceed to purification - Sequentially transfer 1000uL 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 ::: callout-caution Forgot to run DNA/RNA Wash prior to DNase I treatment!!! ::: ```{r} DB = 75*6 print(DB) Dnase = 5*6 print(Dnase) ``` - 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 cnical end of the tube. This worked well and I had a full 80uL of treatment per spin column to work with. - Warmed Zymo DNase-RNase Free Water to 55C in heat block before elution, and slowly dripped water directly over filter. eluted DNA volume: 30uL 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 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: 96.43 standard 2: Error... re-run cal standards? No... RunID: 2024-08-17_045016 | sample_id | qubit_rna_1 | qubit_rna_2 | |-----------|-------------|-------------| | 123L4 | 10.9 ng/uL | 10.9 ng/uL | | 789L9 | 6.32 ng/uL | 6.26 ng/uL | | 101112C9 | 5.56 ng/uL | 5.46 ng/uL | | 101112H9 | 8.46 ng/uL | 8.46 ng/uL | | 131415M14 | 7.38 ng/uL | 7.32 ng/uL | | 131415C4 | 11.1 ng/uL | 11.1 ng/uL | # 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: