Extractions for e5 time series

This protocol is based on the Putnam lab Zymo Miniprep protocol (see example from Zoe here). This post details the info about the extraction steps for 12 samples from the e5 timeseries (Acropora, Pocillopora, Porites spp.) in Moorea 2020. The e5 github is linked here.

Samples

The samples run today were re-extractions from the e5 deep dive samples. We had already sequenced these samples but we are going to sequence them again for the timeseries molecular analysis so we decided to re-extract so there were no batch effects. Since the samples had already been through one extraction, there was <1mL of DNA/RNA shield in the tube.

• 401
• 417
• 423
• 427
• 439
• 491

This photo is before I removed shield from the sample tubes:

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
  • 2 Qubit tubes per sample

Protocol

• 300 uL of sample was moved from the original shield tube into a new 1.5mL tube for the prot-k digestion step.

• For 491, 100 uL of shield + 200 uL of new shield was moved into a new tube. Kristina did this with some of her extractions and it worked well for Porites.

• Samples were snap frozen (in 2020) and clipped into a tube with 1 mL of DNA/RNA shield and 0.5mm glass beads.
• Samples were bead beat for 1.5 minutes on max speed.

This protocol was followed exactly and eluted in 100 uL of either Tris (DNA) or DNA/RNA free water (RNA).

QC

Qubit results

For Qubit, I used the DNA HS kit and the RNA HS kit.

Shield Tube	Colony ID	Timepoint	Volume	DNA1_ng_uL	DNA2_ng_uL	RNA1_ng_uL	RNA2_ng_uL	DNA_average	RNA_average	DNA_ug	RNA_ug	DNA_ng	RNA_ng
401	POC-53	TP2	90	29	28.8	19.9	19.8	28.9	19.85	2.601	1.7865	2601	1786.5
417	POC-57	TP2	90	77	76.2	39.8	39.4	76.6	39.6	6.894	3.564	6894	3564
423	ACR-150	TP2	90	36.8	36.6	14.6	14.7	36.7	14.65	3.303	1.3185	3303	1318.5
427	ACR-145	TP2	90	19.4	19.3	14	13.8	19.35	13.9	1.7415	1.251	1741.5	1251
439	ACR-173	TP2	90	36.4	36.2	8.84	8.72	36.3	8.78	3.267	0.7902	3267	790.2
491	POR-73	TP2	90	1.48	1.47	0	0	1.475	0	0.13275	0	132.75	0

All samples look good for DNA and RNA concentrations except for 491. It had a low DNA concetration and no RNA was detected, even though I used the High Sensitivity kits for both assays.

Gel

I ran a 1.5% gel at 100 volts for 1 hour. Samples are in the same order as the Qubit with an empty well in between the ladder and the samples.

There is DNA there, some degradation. No DNA for 491. No RNA showed up, which was weird. Could be degradation or gel loading issues.