1 Samples

4C14F, 5C14F = 45C14
- 2 min Qiagin Tissue Disruption Tubes
4L4F, 5L4F, 6L4F = 456L4
- 2 min Qiagin Tissue Disruption Tubes
4M9F, 5M9F, 6M9F = 456M9
- 2 min Qiagin Tissue Disruption Tubes
4H4F, 5H4F, 6H4F = 456H4
- 2 min Qiagin Tissue Disruption Tubes
4C9F, 5C9F = 45C9
- 2 min Qiagin Tissue Disruption Tubes
4L9F, 5L9F, 6L9F = 456L9
- 2 min Qiagin Tissue Disruption Tubes

2 Summary

Caution

This extraction was overall bad… The main difference was I re-introduced Proteinase K digestion (hoping to maintain RNA quality & integrity… But RNA quantity suffered A LOT (And I’m not measuring RIN… just crossing my fingers and sending it to Azenta)

3 Extraction Notes

date: 15-AUG-2024

kit: Zymo Quick-DNA/RNA Miniprep Plus Kit

Thaw samples to room temp
Vortex to mix
Transfer to Qiagin Tissue Disruption Tube
Homogenize for 2 minutes
Centrifuge 1 minute to get rid of bubbles
- Checked for complete lysis… still saw entact cells so I homogenized again for 1 minute with 1/3 of Zymo 0.1 & 0.5 mm glass beads poured in each tube. This resulted in complete lysis. Centrifuged again for 1 minute
Split into two aliquots of 500uL & transfered to two new nuclease-free tubes
Proteinase K Digestion for 40 minutes at 55C
Added 700uL of DNA/RNA Lysis Buffer to each tube ( a total of 1400uL volume in each tube )
Vortex to mix for 5 seconds
Check to see that embryos have dissolved and liquid is clear

—> Proceed to purification

Sequentially transfer 2800uL of each sample in aliquots of 700uL into yellow Spin-Away Filter in a Collection Tube and centrifuge for 30s. SAVE THE FLOW THROUGH FOR RNA PURIFICATION by tipping flow through into 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 = 5mL
Sequentially transfer RNA flow-through + ethanol in volumes =>700uL into the green Zymo Spin IIICG Column in a Collection Tube
Saved 1st 1400uL for protein

Warning

This took 7 passes!
Performed DNase I Treatment on green RNA spin column
```
DB = 75*4
print(DB)
```
```
[1] 300
```
```
Dnase = 5*4 
print(Dnase)
```
```
[1] 20
```
- 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: 30uL in Zymo DNase-RNase Free Water

Important

I spoke with Azenta rep on morning of 15-Aug-2024 and learned that they don’t require a Nanodrop quality check, just a Qubit double-stranded quantity measurement by flourescence. The target is 100ng of total RNA, in a minimum of 20uL of buffer. So from here on out I’m just checking quantity using the Qubit and the Invitrogen High Sensitivity (HS) RNA assay.

4 Qubit

Using the Invitrogen Thermo Fischer RNA High Sensitivity Assay Kit.

Prepared working solution for 3 samples and two standards:

199*6

[1] 1194

Qubit RNA HS reagent in DMSO = 1uL * 5 = 6uL

Qubit RNA HS Buffer = 199uL * 5 = 1194uL

4.0.1 RNA

standard 1: 94.63

standard 2: 1803.97

RunID: 2024-08-16_074027

sample_id	qubit_rna_1	qubit_rna_2
45C14	8.70 ng/uL	8.88 ng/uL
456L4	too low	too low
456M9	too low	too low
456H4	too low	too low
45C9	too low	too low
456L9	8.34 ng/uL	8.22 ng/uL

5 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: "15-Aug-2024 Embryo RNA Extractions" author: "Sarah Tanja" date: 2024-08-15 date-format: long date-modified: today categories: [lab records] draft: false image: "samples-in-rack.jpg" --- # Samples - 4C14F, 5C14F = 45C14 - 2 min Qiagin Tissue Disruption Tubes - 4L4F, 5L4F, 6L4F = 456L4 - 2 min Qiagin Tissue Disruption Tubes - 4M9F, 5M9F, 6M9F = 456M9 - 2 min Qiagin Tissue Disruption Tubes - 4H4F, 5H4F, 6H4F = 456H4 - 2 min Qiagin Tissue Disruption Tubes - 4C9F, 5C9F = 45C9 - 2 min Qiagin Tissue Disruption Tubes - 4L9F, 5L9F, 6L9F = 456L9 - 2 min Qiagin Tissue Disruption Tubes # Summary ::: callout-caution This extraction was overall bad... The main difference was I re-introduced Proteinase K digestion (hoping to maintain RNA quality & integrity... But RNA quantity suffered A LOT (And I'm not measuring RIN... just crossing my fingers and sending it to Azenta) ::: # Extraction Notes date: 15-AUG-2024 kit: Zymo Quick-DNA/RNA Miniprep Plus Kit - Thaw samples to room temp - Vortex to mix - Transfer to Qiagin Tissue Disruption Tube - Homogenize for 2 minutes - Centrifuge 1 minute to get rid of bubbles - Checked for complete lysis... still saw entact cells so I homogenized again for 1 minute with 1/3 of Zymo 0.1 & 0.5 mm glass beads poured in each tube. This resulted in complete lysis. Centrifuged again for 1 minute - Split into two aliquots of 500uL & transfered to two new nuclease-free tubes - Proteinase K Digestion for 40 minutes at 55C - Added 700uL of DNA/RNA Lysis Buffer to each tube ( a total of 1400uL volume in each tube ) - Vortex to mix for 5 seconds - Check to see that embryos have dissolved and liquid is clear ---\> Proceed to purification - Sequentially transfer 2800uL of each sample in aliquots of 700uL into yellow Spin-Away Filter in a Collection Tube and centrifuge for 30s. **SAVE THE FLOW THROUGH FOR RNA PURIFICATION by tipping flow through into 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 = 5mL - Sequentially transfer RNA flow-through + ethanol in volumes =\>700uL into the green Zymo Spin IIICG Column in a Collection Tube - Saved 1st 1400uL for protein ::: callout-warning This took 7 passes! ::: - Performed DNase I Treatment on green RNA spin column ```{r} DB = 75*4 print(DB) Dnase = 5*4 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: 30uL in `Zymo DNase-RNase Free Water` ::: callout-important I spoke with Azenta rep on morning of 15-Aug-2024 and learned that they don't require a Nanodrop quality check, just a Qubit double-stranded quantity measurement by flourescence. The target is 100ng of total RNA, in a minimum of 20uL of buffer. So from here on out I'm just checking quantity using the Qubit and the Invitrogen High Sensitivity (HS) RNA assay. ::: # Qubit Using the Invitrogen Thermo Fischer RNA High Sensitivity Assay Kit. Prepared working solution for 3 samples and two standards: ```{r} 199*6 ``` Qubit RNA HS reagent in DMSO = 1uL \* 5 = 6uL Qubit RNA HS Buffer = 199uL \* 5 = 1194uL ### RNA standard 1: 94.63 standard 2: 1803.97 RunID: 2024-08-16_074027 | sample_id | qubit_rna_1 | qubit_rna_2 | |-----------|-------------|-------------| | 45C14 | 8.70 ng/uL | 8.88 ng/uL | | 456L4 | too low | too low | | 456M9 | too low | too low | | 456H4 | too low | too low | | 45C9 | too low | too low | | 456L9 | 8.34 ng/uL | 8.22 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: