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

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

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.

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

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: