Soft and Hard Homogenization DNA/RNA Extractions #267-282

20190805 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).
• 2 coral fragments were used twice (6 coral fragments, 2 of which were done twice. 8 bead tubes used total) to compare DNA quality after the addition of a RNA/DNA shield wash. 500 μl of RNA/DNA shield “wash” step added before the 700 μl of Wash Buffer step for the duplicate extractions.

Coral fragments were randomly chosen from different timepoint bags.

Fragment preparation start 11:00 end 11:18
Soft/Hard homogenization start 11:19 end 11:51
RNA DNA Extraction start 11:52 end 14:42

Extraction #s: 269, 270, 277, and 278 got the 500 μl RNA/DNA shield “wash” step in the DNA extractions only.

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

DNA Standard 1: 190.13 ng/μl
DNA Standard 2: 21,227.88 ng/μl
RNA Standard 1: 397.54 ng/μl
RNA Standard 2: 10,498.84 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
20180929	Pocillopora	1721	267	soft	34	33.8	33.9	80.4	79.4	79.9	7.6
20180929	Pocillopora	1721	268	hard	19.4	19.3	19.35	54.8	54.8	54.8	7.6
20180929	Pocillopora	1721	269	soft	41.4	41.4	41.4	123	123	123	8
20180929	Pocillopora	1721	270	hard	24.6	24.6	24.6	86.2	85.8	86	8
20180929	Montipora	1114	271	soft	24.8	24.8	24.8	36	36	36	8.7
20180929	Montipora	1114	272	hard	10.1	10	10.05	23.6	23.6	23.6	6.4
20180922	Pocillopora	2527	273	soft	68.2	67.8	68	125	125	125	7.7
20180922	Pocillopora	2527	274	hard	61.2	61.2	61.2	87.2	87.2	87.2	8.2
20180922	Pocillopora	1637	275	soft	86.6	86.6	86.6	106	106	106	7.9
20180922	Pocillopora	1637	276	hard	49.6	49.6	49.6	69.4	69.4	69.4	7.7
20180922	Pocillopora	1637	277	soft	38	37.8	37.9	82	82	82	8
20180922	Pocillopora	1637	278	hard	19.7	19.6	19.65	63.8	64	63.9	**
20181020	Montipora	1082	279	soft	12.9	12.8	12.85	14	14	14	**
20181020	Montipora	1082	280	hard	9.18	9.1	9.14	12.2	12.2	12.2	8.2
20180929	Montipora	1212	281	soft	9.96	9.94	9.95	10.6	10.6	10.6	6.2
20180929	Montipora	1212	282	hard	7.58	7.52	7.55	**	**	**	7.6

Think about re-qubiting 1212 hard 282 and re-tapestation 1637 hard 278.

Link to 20190805 TapeStation report, Extractions #267-282

Gel Electrophoresis Results: