Dual DNA & RNA Extraction #1 (ex1)

Notebook post detailing lab-work conducted on April 25th, 2023 (4/25/2023)

This lab extraction follows the Dual DNA & RNA Extraction Protocol with any deviations, exceptions, or notes explained in detail.

Samples

• 3-CA1a

• 3-Ea

Samples were randomly selected for processing given constraints explained in this RMarkdown document.

Metadata and notes are also recorded in the extr1_21APR23.csv

Key Results

cryo_id	extr_date	qubit_rna_run_date	qubit_rna_1	qubit_rna_2	qubit_rna3	qubit_dna_run_date	qubit_dna_1	qubit_dna_2	qubit_dna_3
3-CA1a	4/21/2023	4/25/2023	12.4	14	12.4	5/4/2023	72.8	75	74.4
3-Ea	4/21/2023	4/25/2023	0.0	0	0	5/4/2023	39	38.4	39

• qubit_rna_1 was run by SST and NAH at 1140 on 25APR2023, Run ID 2023-04-25-110503

• qubit_rna_2 and qubit_rna_3 were run on 2 separately prepared technical replicates by SST at 1340 on 25APR2023, Run ID 2023-04-25-133744

• exported RNA Qubit Data file (Run ID 2023-04-25-133744 & 2023-04-25-110503) with more details can be found HERE

• qubit_dna was checked on 5/4/2023 because we were waiting for the Thermo Qubit DNA Broad Range (BR) Assay Kit to be delivered. Samples were frozen in a -80C freezer between extraction and being checked by Qubit for quantity

• exported DNA Qubit Data file (Run ID 2023-05-02-102849) with more details can be found HERE

Samples are stored in -80C ‘sucks’ freezer in respective Eluted-DNA or Eluted-RNA wax freezer boxes with the labels:

Freezer	Wax-Box	Vial
-80C sucks	Eluted-DNA	3CA1a DNA extr1 21APR23 NAH + SST
-80C sucks	Eluted-DNA	3Ea DNA extr1 21APR23 NAH + SST
-80C sucks	Eluted-RNA	3CA1a RNA extr1 21APR23 NAH + SST
-80C sucks	Eluted-RNA	3Ea RNA extr1 21APR23 NAH + SST

Sterilized Lab Bench

• used RNAse away only on gloves and items that were touching the sample (scoopula, mortar, pestle, forceps)

Labelled Tubes

cryo_ID’s :

• 3-CA1a

• 3Ea

Prepared Buffers

• Used instruction for the 10-prep kit, (D7003T)
• Used buffers fresh, stored extra DNase 1 and Proteinase K in -20C after use on 4/25/2023

Extraction Steps

Pestled Samples

• 3-CA1a pestled 1st, half of the material in the cryo vial was used. Half remains in the freezer.
• 3-Ea pestled 2nd, all of the material in the cryo vial was used, but half fell on the floor and had to be thrown out.
• LN2 evaporates very quickly and had to be replenished multiple times
• We did this best with two people (NAH to grind, SST to carefully dispense LN2)

⚠️Warning⚠️: The coral is very hard to pestle and prone to ‘squirting out’ from under the pestle, much like the gif below! Learned that it is better to leverage whole body weight above the mortar and work to crush the coral fragment like a hydraulic press. When ‘attacking’ sample 3-Ea at an angle, half of the material from shot out of the mortar and ended up on the floor, resulting in less starting material left to use in the mortar. This could be a reason why 3-Ea had ‘out of range, too low’ result for Qubit! {: .notice–warning}

• Use sterilized scoopula to transfer the sample powder to its correspondingly labelled bead bashing tube.

For this step we made small disposable funnels from lab weigh-paper and label tape to try and make this easier. Unfortunately, ⚠️this didn’t work very well! See warning below…

⚠️Warning⚠️: Finely ground powder melts fast and the cold from the LN2 means that all the moisture in the lab is condensing on the sample. As soon as it is removed from the mortar by the scoopula it begins to melt, condense water vapor, and turn into a sticky booger! It doesn’t slide nice and dry down the paper funnel. We had to tamp it vigorously to get it into the bead bashing tube. We will modify protocol next time to first chill the scoopulas on dry-ice, and use a small metal funnel chilled on dry-ice, with the goal being to keep the powder cold during the transfer from the mortar to inside the bead bashing tube. {: .notice–warning}

• Once sample powder was in the bead bashing tube, we quickly added 750uL of DNA/RNA Sheild to the bead bashing tube, shook it vigorously & vortexed it to ensure the sample was submersed in DNA/RNA Sheild

⚠️Warning⚠️: In hindsight this was too much volume for the bead bashing tube! After bead beating tube was very very bubbly! Would suggest centrifuging after the bead-beating homogenization ‘shaker’ step prior to opening tube lid and/or use 500uL of DNA/RNA Sheild, and putting it in the bead bashing tube before the sample in future extractions. {: .notice–warning}

• We then set bead-bashing tubes in the Mortexer and let it mix at max speed for 40 mins

Proteinase-K Digestion

• First added 30uL of Proteinase-K Digestion Buffer directly to the bead bashing tube
• Then added 15uL of Proteinase-K directly to the bead bashing tube
• Vortexed to mix
• Incubated (left to sit) at room temp for 30 minutes

##

• Centrifuged at max-speed (16,160xg) for 30 seconds

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Purify DNA & RNA

End Products

The end products are two 1.5mL vials per sample that contain:

1. 100ul of DNA in nuclease-free water
2. 100ul of RNA in nuclease-free water.

Place these vials on ice and for RNA proceed with Qubit RNA Broad Range Protocol , for DNA proceed with Nanodrop DNA

OR, to continue lab-work later,

Place them in a wax freezer box, label the box, and freeze them in the -80C.