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

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

Danger

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.

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

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: