September DNA RNA Extractions

20190903 E.S.

DNA/RNA Extractions from Montipora capitata and Pocillopora acuta adult coral fragments from Holobiont Integration Hawaii 2018 project.

Soft and Hard homogenization, and DNA/RNA Extractions followed this protocol. General Zymo Duet DNA/RNA Extraction protocol found here. With the following changes:

• DNA: The first and second elutions were spun down in separate microcentrifuge tubes to compare the quantity and quality between the first and second elution. The first elution had no incubation period (previously 5 minutes) with 10 μl of Tris buffer and the second elution had a 15 minute incubation period (previously 5 minutes) with 100 μl of Tris buffer.
• 10 μl aliquots of DNA and 5 μl aliquots of RNA (instead of 10 μl of each).

Coral fragments were randomly chosen from different timepoint bags.

Ran out of wash buffer during RNA extractions and was only able to complete the full RNA extraction for samples #418, 419, 421, and 422.

Qubit used to check DNA and RNA quantity (ng/μl).
TapeStation used to check RNA quality.

DNA Standard 1: 207.83 ng/μl
DNA Standard 2: 22,361.36 ng/μl
RNA Standard 1: 389.41 ng/μl
RNA Standard 2: 9,965.77 ng/μl

Timepoint	Species	Coral ID	Extraction ID	Homogenization	DNA Reading 1	DNA Reading 2	Average DNA ng/μl	RNA Reading 1	RNA Reading 2	Average RNA ng/μl	RIN
20180922	Montipora	1548	417	soft	30.2	30	30.1	NA	NA	NA	NA
20180922	Montipora	1548	418	hard	12.5	12.4	12.45	17.2	17	17.1	NA
20180922	Montipora	1628	419	soft	35.6	35.4	35.5	46	46	46	8.9
20180922	Montipora	1628	420	hard	21.2	21.2	21.2	NA	NA	NA	NA
20180922	Pocillopora	1642	421	soft	46	46	46	105	105	105	8.5
20180922	Pocillopora	1642	422	hard	40	39.8	39.9	61.2	61.2	61.2	NA
20180922	Pocillopora	2750	423	soft	64.2	64	64.1	NA	NA	NA	NA
20180922	Pocillopora	2750	424	hard	44.2	44	44.1	NA	NA	NA	NA
20180923	Montipora	1449	425	soft	18.7	18.6	18.65	NA	NA	NA	NA
20180923	Montipora	1449	426	hard	15.3	15.3	15.3	NA	NA	NA	NA
20180923	Pocillopora	1563	427	soft	21.6	21.4	21.5	NA	NA	NA	NA
20180923	Pocillopora	1563	428	hard	24.8	24.8	24.8	NA	NA	NA	NA
20180923	Montipora	1997	429	soft	18.2	18.1	18.15	NA	NA	NA	NA
20180923	Montipora	1997	430	hard	10.5	10.5	10.5	NA	NA	NA	NA
20180923	Montipora	1694	431	soft	15.1	15	15.05	NA	NA	NA	NA
20180923	Montipora	1694	432	hard	13.2	13.2	13.2	NA	NA	NA	NA
20180923	Pocillopora	2212	433	soft	47.6	47.4	47.5	NA	NA	NA	NA
20180923	Pocillopora	2212	434	hard	24.2	24	24.1	NA	NA	NA	NA
20180923	Pocillopora	1707	435	soft	30.8	30.8	30.8	NA	NA	NA	NA
20180923	Pocillopora	1707	436	hard	35.6	35.6	35.6	NA	NA	NA	NA

Link to 20190903 TapeStation report, Extractions #417-436

Gel Electrophoresis Results:

20190904 E.S. Extraction

Coral fragments #1548 and #1628 re-extracted because not enough wash buffer during yesterday’s extractions to complete the set. See 20190903 description for links to protocols used.

Timepoint	Species	Coral ID	Extraction ID	Homogenization	DNA Reading 1	DNA Reading 2	Average DNA ng/μl	RNA Reading 1	RNA Reading 2	Average RNA ng/μl	RIN
20180922	Montipora	1548	437	soft	23.2	23	23.1	24.2	24.2	24.2	8
20180922	Montipora	1548	438	hard	16.9	16.9	16.9	19.8	19.8	19.8	NA
20180922	Montipora	1628	439	soft	17	17	17	28.2	28.2	28.2	8.7
20180922	Montipora	1628	440	hard	21.8	21.6	21.7	24	24	24	NA

DNA Standard 1: 207.83 ng/μl
DNA Standard 2: 22,361.36 ng/μl
RNA Standard 1: 389.41 ng/μl
RNA Standard 2: 9,965.77 ng/μl

Link to 20190904 TapeStation report, Extractions #437-440

Gel Electrophoresis Results:

20190905 E.S. Extraction

The eight coral fragments that were not re-extracted on 20190904 were extracted on 20190905 because not enough wash buffer during 20190903’s extractions to complete the set. See 20190903 description for links to protocols used.

Fragment preparation start 12:49 end 13:09
Soft/Hard homogenization start 13:10 end 13:38
RNA DNA Extraction start 13:39 end 15:45

DNA Standard 1: 189.79 ng/μl
DNA Standard 2: 21,084.91 ng/μl
RNA Standard 1: 399.67 ng/μl
RNA Standard 2: 9,715.01 ng/μl

Timepoint	Species	Coral ID	Extraction ID	Homogenization	DNA Reading 1	DNA Reading 2	Average DNA ng/μl	RNA Reading 1	RNA Reading 2	Average RNA ng/μl	RIN
20180923	Montipora	1449	441	soft	27.2	27	27.1	59.6	59.6	59.6	8.7
20180923	Montipora	1449	442	hard	22.2	22.2	22.2	26.8	26.8	26.8	NA
20180922	Pocillopora	1642	443	soft	70	70	70	80.2	80.2	80.2	7.5
20180922	Pocillopora	1642	444	hard	37.4	37.2	37.3	40	40.2	40.1	NA
20180923	Pocillopora	1707	445	soft	96.6	96.4	96.5	77.4	77.6	77.5	8.5
20180923	Pocillopora	1707	446	hard	35.4	35.4	35.4	42.2	42.4	42.3	NA
20180923	Pocillopora	1563	447	soft	42.4	42.2	42.3	39.8	39.8	39.8	8.7
20180923	Pocillopora	1563	448	hard	24	24	24	33.2	33.2	33.2	NA
20180923	Pocillopora	2212	449	soft	34.2	34.2	34.2	54	53.8	53.9	8.1
20180923	Pocillopora	2212	450	hard	19.3	19.3	19.3	43.6	43.6	43.6	NA
20180923	Montipora	1694	451	soft	19.2	19.2	19.2	25	25.2	25.1	9
20180923	Montipora	1694	452	hard	14.6	14.5	14.55	16.6	16.6	16.6	NA
20180923	Montipora	1997	453	soft	23.4	23	23.2	**	**	**	9.1
20180923	Montipora	1997	454	hard	11.2	11.2	11.2	**	**	**	NA
20180922	Pocillopora	2750	455	soft	60.6	60.6	60.6	85.4	85.4	85.4	8.6
20180922	Pocillopora	2750	456	hard	45.8	45.8	45.8	45.2	45	45.1	NA

Extraction #453 and #454 read “Too Low” but the TapeStation report a 9.1 value for #453. Think about re-qubitting #453 and #454.

Link to 20190905 TapeStation report, Extractions #441-456

Gel Electrophoresis Results:

20190924 E.S.

DNA/RNA Extractions from Montipora capitata and Pocillopora acuta adult coral fragments from Holobiont Integration Hawaii 2018 project.

Soft and Hard homogenization, and DNA/RNA Extractions followed this protocol. General Zymo Duet DNA/RNA Extraction protocol found here. With the following changes:

• DNA: The first and second elutions were spun down in separate microcentrifuge tubes to compare the quantity and quality between the first and second elution. The first elution had no incubation period (previously 5 minutes) with 10 μl of Tris buffer and the second elution had a 15 minute incubation period (previously 5 minutes) with 100 μl of Tris buffer.
• 10 μl aliquots of DNA and 5 μl aliquots of RNA (instead of 10 μl of each).

M1236 was very dark brown, almost black and smelled bad. Much darker than the rest of the Montipora fragments.

30 mL of Puritz’s ethanol used. Replaced 10.5 mL of Puritz Wash Buffer.

Qubit used to check DNA and RNA quantity (ng/μl).
TapeStation used to check RNA quality.

DNA Standard 1: 131.30 ng/μl
DNA Standard 2: 18,106.18 ng/μl
RNA Standard 1: 400.44 ng/μl
RNA Standard 2: 10,114.91 ng/μl

Timepoint	Species	Coral ID	Extraction ID	Homogenization	DNA Reading 1	DNA Reading 2	Average DNA ng/μl	RNA Reading 1	RNA Reading 2	Average RNA ng/μl	RIN
20181006	Pocillopora	1281	457	soft	34.2	34	34.1	64.4	64.4	64.4	8.4
20181006	Pocillopora	1281	458	hard	16.7	16.7	16.7	39.4	39.4	39.4	NA
20180922	Montipora	1651	459	soft	24.6	24.4	24.5	56.6	56.8	56.7	9.1
20180922	Montipora	1651	460	hard	27	27	27	31.2	31.4	31.3	NA
20180922	Montipora	1236	461	soft	**	**	**	17.8	17.8	17.8	2.7
20180922	Montipora	1236	462	hard	**	**	**	13.6	13.8	13.7	NA
20180923	Montipora	1229	463	soft	4.56	4.48	4.52	14.2	14.4	14.3	8.9
20180923	Montipora	1229	464	hard	3.64	3.6	3.62	15.6	15.6	15.6	NA

20190925 E.C. D.C.

Link to 20190925 TapeStation report, Extractions #457-464

Gel Electrophoresis Results:

20190926 E.S.

DNA/RNA Extractions from Montipora capitata and Pocillopora acuta adult coral fragments from Holobiont Integration Hawaii 2018 project.

Soft and Hard homogenization, and DNA/RNA Extractions followed this protocol. General Zymo Duet DNA/RNA Extraction protocol found here. With the following changes:

• DNA: The first and second elutions were spun down in separate microcentrifuge tubes to compare the quantity and quality between the first and second elution. The first elution had no incubation period (previously 5 minutes) with 10 μl of Tris buffer and the second elution had a 15 minute incubation period (previously 5 minutes) with 100 μl of Tris buffer.
• 10 μl aliquots of DNA and 5 μl aliquots of RNA (instead of 10 μl of each).

P1582 fragment is bleached completely but no algae on the tips of the fragment.

Qubit used to check DNA and RNA quantity (ng/μl).
TapeStation used to check RNA quality.

DNA Standard 1: 159.23 ng/μl
DNA Standard 2: 22,818.49 ng/μl
RNA Standard 1: 404.22 ng/μl
RNA Standard 2: 9,965.62 ng/μl

Timepoint	Species	Coral ID	Extraction ID	Homogenization	DNA Reading 1	DNA Reading 2	Average DNA ng/μl	RNA Reading 1	RNA Reading 2	Average RNA ng/μl	RIN
20181215	Pocillopora	2306	465	soft	73.4	72.8	73.1	77.2	77.2	77.2	8.4
20181215	Pocillopora	2306	466	hard	54	53.6	53.8	46.4	46.4	46.4	7.8
20181215	Montipora	1562	467	soft	29.4	29.4	29.4	34.2	34.2	34.2	8.3
20181215	Montipora	1562	468	hard	18.4	18.3	18.35	20	20	20	8.6
20181215	Pocillopora	1647	469	soft	98.4	97.8	98.1	96.8	96.8	96.8	7.6
20181215	Pocillopora	1647	470	hard	59.4	59.2	59.3	57.4	57.4	57.4	7.5
20181215	Montipora	1237	471	soft	32	31.8	31.9	32.2	32.2	32.2	8.4
20181215	Montipora	1237	472	hard	17.6	17.4	17.5	18.4	18.4	18.4	8.3
20181215	Pocillopora	1582	473	soft	34.8	34.6	34.7	78.8	78.6	78.7	8.9
20181215	Pocillopora	1582	474	hard	21	20.8	20.9	47.6	47.6	47.6	8.6

Link to 20190926 TapeStation report, Extractions #465-474

Gel Electrophoresis Results:

20190930 E.S.

DNA/RNA Extractions from Montipora capitata and Pocillopora acuta adult coral fragments from Holobiont Integration Hawaii 2018 project.

Soft and Hard homogenization, and DNA/RNA Extractions followed this protocol. General Zymo Duet DNA/RNA Extraction protocol found here. With the following changes:

• DNA: The first and second elutions were spun down in separate microcentrifuge tubes to compare the quantity and quality between the first and second elution. The first elution had no incubation period (previously 5 minutes) with 10 μl of Tris buffer and the second elution had a 15 minute incubation period (previously 5 minutes) with 100 μl of Tris buffer.
• 10 μl aliquots of DNA and 5 μl aliquots of RNA (instead of 10 μl of each).

Montipora samples were atypically vortexed for 1 minute instead of 2 minutes during the soft homogenization step.

Qubit used to check DNA and RNA quantity (ng/μl).
TapeStation used to check RNA quality.

RNA Qubit Results: 20191001
DNA Qubit Results: 20191002

DNA Standard 1: 149.59 ng/μl
DNA Standard 2: 20,884.81 ng/μl
RNA Standard 1: 404.99 ng/μl
RNA Standard 2: 10,128.77 ng/μl

Timepoint	Species	Coral ID	Extraction ID	Homogenization	DNA Reading 1	DNA Reading 2	Average DNA ng/μl	RNA Reading 1	RNA Reading 2	Average RNA ng/μl	RIN
20181215	Montipora	2380	475	soft	15.2	15.2	15.2	24.2	24.2	24.2	9
20181215	Montipora	2380	476	hard	7.54	7.5	7.52	19.4	19.4	19.4	**
20181215	Montipora	1178	477	soft	11.9	11.9	11.9	24.6	24.6	24.6	**
20181215	Montipora	1178	478	hard	14.6	14.5	14.55	18.8	18.8	18.8	9.2
20181215	Montipora	1076	479	soft	19	18.8	18.9	22.2	22.2	22.2	7.6
20181215	Montipora	1076	480	hard	13.5	13.3	13.4	17.2	17.2	17.2	**
20181215	Pocillopora	1777	481	soft	46.6	46.4	46.5	65.8	65.8	65.8	7.8
20181215	Pocillopora	1777	482	hard	30	29.8	29.9	32.4	32.6	32.5	7.3
20181215	Pocillopora	2879	483	soft	43.2	43	43.1	94	94.2	94.1	8.4
20181215	Pocillopora	2879	484	hard	35.8	35.6	35.7	57.4	57.6	57.5	8.1

Link to 20190930 TapeStation report, Extractions #475-484

Gel Electrophoresis Results: