Extractions for Astrangia 2021 experiment

This protocol is based on those from E. Strand, K. Wong, E. Chille, K. Terpis, & Z. Delleart.

Samples

Samples that I used today:

• AST-2007
• AST-1692
• AST-2729
• AST-1105
• AST-1412
• AST-2404
• AST-2302
• AST-2412

Samples were flash frozen, clipped, and put in 1.5 mL bead tube in 1 mL of DNA/RNA shield. AST-2007 is pretty cloudy. I couldn’t find the original tube with the fragment in it. I found a 1.5 mL tube labeled 2007 with homogenate in DNA/RNA shield in the -20°C freezer. I believe I used AST-2007 when I trialed the Mag-Bead kit in March 2022, but the kit was unsuccessful. See post here.

This photo is post bead beating:

Materials

• Zymo Quick-DNA/RNA Miniprep Plus Kit HERE Protocol Booklet
• Tris-Ethylenediaminetetraacetic acid (EDTA) 1X buffer for DNA elution
• Heating block capable of heating to 70ºC
• Centrifuge and rotor capable of spinning at 15,000 rcf
• Plastics
  • 5 1.5 mL microcentrifuge tubes per sample
  • 2 PCR tubes per sample
  • Several 1000 uL, 200 uL, and 20 uL filtered pipette tips per sample.

Protocol

Before extractions, I bead beat samples, as they were already in bead tubes

• Bead beat for 1 minute at max speed
• Quick spin down in smol centrifuge
• Transfer 400 μL into new tube.
• Centrifuge for 3 minutes at 9000 rct
• Transfer 300 μL into new tube

Used this protocol (mine) for extractions and QC.

Quality Control

Qubit results

Tube	Type	DNA 1 (ng/uL)	DNA 2 (ng/uL)	RNA 1 (ng/uL)	RNA 2 (ng/uL)
S1	Standard	190.54	NA	392.12	NA
S2	Standard	22388.23	NA	7956.48	NA
AST-2007	Sample	10.8	10.9	23	23
AST-1692	Sample	2	2.02	NA	NA
AST-2729	Sample	11.5	11.6	22	21.4
AST-1105	Sample	47	48.2	53.6	54.2
AST-1412	Sample	19	19.1	44.6	45
AST-2404	Sample	7.68	7.92	26	26.2
AST-2302	Sample	43	43.2	55	55
AST-2412	Sample	7.98	8.06	16.6	16.8

Gel

I messed up the gel the first time so I had to rerun it. I took 9ul from the 90ul stock so these samples only have ~81ul left.