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 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 and timeseries samples. I am having library prep issues with 6 of the deep dive samples and ~29 of the timeseries DNA samples do not have enough ng of DNA for Azenta. I am going to re-extract to see if we can get higher concentrations. Since the samples had already been through one extraction, there was <1mL of DNA/RNA shield in the tube.

Sample	tube #	Purpose	Notes
7-20220208	7	timeseries
13	13	timeseries
23	23	timeseries
29	29	timeseries
195	195	timeseries
20240805_POC-52_TP1	213	timeseries	Might be okay with original DNA?
235	235	timeseries
247-20220208	247	timeseries
251-20220208	251	timeseries
297	297	timeseries
319	319	timeseries
327	327	timeseries
385	385	deepdive
393	393	deepdive
401	401	deepdive
421	421	deepdive
469-20211122	469	timeseries
477	477	timeseries
487	487	deepdive
489	489	deepdive
491	491	timeseries
493	493	timeseries
515-20211104	515	timeseries
20240805_POC-219_TP3	521	timeseries	Might be okay with original DNA?
529	529	timeseries
557	557	timeseries
633-20220201	633	timeseries
637	637	timeseries
641	641	timeseries
669	669	timeseries
711	711	timeseries
713	713	timeseries
741	741	timeseries
749	749	timeseries
877	877	timeseries

There were 35 samples total, so I split them into two batches because the centrifuge can only fit 24 tubes at a time. 213, 385, 393, 401, and 521 were POC, the rest of the samples were POR.

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

Set 1

Set 2

Materials

ONLY DID DNA EXTRACTIONS

• 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
  • 3 1.5 mL microcentrifuge tubes per sample
  • 1 PCR tubes per sample
  • 1 Qubit tubes per sample

Protocol

This protocol was followed. A few differences

• For POC samples, 300 uL of sample was moved from the original shield tube into a new 1.5mL tube for the prot-k digestion step.
• For POR samples, 100 uL of sample and 200 uL of fresh DNA/RNA shield was moved into a new 1.5mL tube for the prot-k digestion step.
• Samples were eluted in 90 uL of Tris instead of 100 uL.

QC

Qubit results

For Qubit, I used the DNA HS kit on most samples; on 385, 393, and 401, I used the DNA BR kit because these were out of range with the HS kit. I used 5 uL of DNA as input for the HS kit and 2 uL of DNA as input for the BR kit.

tube #	Colony ID	Time point	Species	Purpose	DNA1_ng_uL	DNA2_ng_uL	DNA_Avg	Volume_uL	DNA_ng
7	POR-262	TP1	Porites evermanni	timeseries	0.39	0.38	0.385	76	29.26
13	POR-245	TP1	Porites evermanni	timeseries	0.386	0.381	0.3835	76	29.146
23	POR-236	TP1	Porites evermanni	timeseries	0.432	0.436	0.434	76	32.984
29	POR-216	TP1	Porites evermanni	timeseries	0.9	0.892	0.896	76	68.096
195	POR-69	TP1	Porites evermanni	timeseries	0.317	0.319	0.318	76	24.168
213	POC-52	TP1	Pocillopora tuahiniensis	timeseries	5.713	5.68	5.6965	76	432.934
235	POR-72	TP1	Porites evermanni	timeseries	0.584	0.23	0.407	76	30.932
247	POR-74	TP1	Porites evermanni	timeseries	0.183	0.19	0.1865	76	14.174
251	POR-73	TP1	Porites evermanni	timeseries	0.193	0.193	0.193	76	14.668
297	POR-245	TP2	Porites evermanni	timeseries	1.28	1.29	1.285	76	97.66
319	POR-236	TP2	Porites evermanni	timeseries	2	2	2	76	152
327	POR-260	TP2	Porites evermanni	timeseries	0.566	0.566	0.566	76	43.016
385	POC-47	TP2	Pocillopora tuahiniensis	deepdive	26.4	25.6	26	74	1924
393	POC-50	TP2	Pocillopora tuahiniensis	deepdive	21.9	21.2	21.55	74	1594.7
401	POC-53	TP2	Pocillopora tuahiniensis	deepdive	26	25.4	25.7	74	1901.8
421	POR-82	TP2	Porites evermanni	deepdive	0.924	0.908	0.916	76	69.616
469	POR-83	TP2	Porites evermanni	timeseries	0.52	0.52	0.52	76	39.52
477	POR-69	TP2	Porites evermanni	timeseries	0.44	0.448	0.444	76	33.744
487	POR-71	TP2	Porites evermanni	deepdive	1.71	1.7	1.705	76	129.58
489	POR-79	TP2	Porites evermanni	deepdive	1.4	1.39	1.395	76	106.02
491	POR-73	TP2	Porites evermanni	timeseries	0.604	0.588	0.596	76	45.296
493	POR-72	TP2	Porites evermanni	timeseries	0.712	0.712	0.712	76	54.112
515	POR-216	TP3	Porites evermanni	timeseries	0.48	0.48	0.48	76	36.48
521	POC-219	TP3	Pocillopora tuahiniensis	timeseries	2.71	2.7	2.705	76	205.58
529	POR-262	TP3	Porites evermanni	timeseries	0.54	0.521	0.5305	76	40.318
557	POR-260	TP3	Porites evermanni	timeseries	0.464	0.444	0.454	76	34.504
633	POR-73	TP3	Porites evermanni	timeseries	1.75	1.74	1.745	76	132.62
637	POR-72	TP3	Porites evermanni	timeseries	0.52	0.516	0.518	76	39.368
641	POR-74	TP3	Porites evermanni	timeseries	0.484	0.48	0.482	76	36.632
669	POR-83	TP3	Porites evermanni	timeseries	0.296	0.297	0.2965	76	22.534
711	POR-83	TP4	Porites evermanni	timeseries	0.298	0.301	0.2995	76	22.762
713	POR-69	TP4	Porites evermanni	timeseries	0.792	0.784	0.788	76	59.888
741	POR-72	TP4	Porites evermanni	timeseries	0.832	0.828	0.83	76	63.08
749	POR-74	TP4	Porites evermanni	timeseries	0.66	0.648	0.654	76	49.704
877	POR-262	TP4	Porites evermanni	timeseries	0.924	0.92	0.922	76	70.072

The POC all look pretty good, but POR is still very low. The highest DNA ng POR value is 152.

Gel

I ran a 1.5% gel at 100 volts for 1 hour. Samples are in the same order as the Qubit.

Gel looks terrible, can’t see anything. There are some faint bands for 385, 393, and 401 (all POC). No bands for POR.