= 75*8
DB print(DB)
[1] 600
= 5*8
Dnase print(Dnase)
[1] 40
Sarah Tanja
August 8, 2025
December 3, 2024
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
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.
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
Centrifuge at 16,000xg for 1 min to get rid of bubbles
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
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!
eluted DNA volume: 50uL in Zymo DNase-RNase Free Water
eluted RNA volume: 50uL in warmed (55C) Zymo DNase-RNase Free Water
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.
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:
standard 1: 92.70
standard 2: 1770.25
RunID: 2024-08-21_054905
sample_id | qubit_rna_1 | qubit_rna_2 |
---|---|---|
|
6.66 ng/uL | 6.68 ng/uL |
|
9.60 ng/uL | 9.52 ng/uL |
|
8.18 ng/uL | 8.18 ng/uL |
|
10.6 ng/uL | 10.4 ng/uL |
|
15.4 ng/uL | 15.4 ng/uL |
|
7.20 ng/uL | 7.02 ng/uL |
|
10.7 ng/uL | 10.8 ng/uL |
|
11.4 ng/uL | 11.4 ng/uL |
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: