During my visit to URI this past week I processed DNA for Sanger sequencing for species identification. These samples are from the 2023 Moorea Symbiotic Exchange project. In this project, I collected corals of three genera (Acropora, Pocillopora, Porites) including adults and recruits. We used Sanger sequencing and RFLP to identify species within these genera.

Overview

gDNA extractions were completed by Pierrick in October 2024.

My samples were included on Plate 38, 39, 40, and 41 of extractions. Sample metadata is on GitHub here.

Protocols used

• gDNA extraction
• Pocillopora mtORF PCR
• Pocillopora Histone PCR and RFLP
• Porites H2 PCR
• Gel electrophoresis
• PCR product cleaning
• Sanger sequencing at URI

Sequencing metadata sheets and all images of gels and associated data is on my GitHub here.

Pocillopora

To identify cryptic species in Pocillopora we use mtORF sequencing as described in Johnston et al. 2018 and Burgess et al. 2024. Our protocol for this is described here.

mtORF sequencing identifies haplotypes. For haplotype 1a (grandis/meandrina complex), we then conduct an RFLP analysis as described in Johnston et al. 2018 to distinguish between these two species. This week I finished up preparing samples for sequencing and ran an RFLP.

mtORF sequencing

20241117

Today I prepared the following samples for mtORF sequencing. These were previously extracted by Pierrick, amplified for mtORF, and the PCR product was cleaned. The plate maps and gels for these samples can be found on GitHub here.

• All plate 39 Huffmyer Pocillopora samples: Wells A1-C8
• Samples in wells F2, F5, F8, F11, and H11 in plate 38 that failed sequencing in a previous run

I’ll post another notebook post detailing sequencing analyses.

Protocol:

1. Dilute forward primer (FatP6.1) to 3.2 uM by adding 33.3 uL of 10 uM primer to 66.6 uL of nuclease-free water
2. Add 8 uL of water to each strip tube for samples
3. Add 2 uL of sample clean PCR product to respective tube
4. Add 2 uL of F primer at 3.2 uM to each tube

Sample information is as follows for samples labeled with “Project 1” for sequencer.

Sample ID	Sample Name	Template Type	PCR Template Volume (µl)	H20 Volume (µl)	Primer Volume (µl)	Primer Name	Notes
AH001	183	PCR	2	8	2	FatP6.1	Strip tubes; water and primer already added
AH002	184	PCR	2	8	2	FatP6.1
AH003	185	PCR	2	8	2	FatP6.1
AH004	186	PCR	2	8	2	FatP6.1
AH005	187	PCR	2	8	2	FatP6.1
AH006	188	PCR	2	8	2	FatP6.1
AH007	189	PCR	2	8	2	FatP6.1
AH008	190	PCR	2	8	2	FatP6.1
AH009	191	PCR	2	8	2	FatP6.1
AH010	194	PCR	2	8	2	FatP6.1
AH011	195	PCR	2	8	2	FatP6.1
AH012	196	PCR	2	8	2	FatP6.1
AH013	197	PCR	2	8	2	FatP6.1
AH014	198	PCR	2	8	2	FatP6.1
AH015	199	PCR	2	8	2	FatP6.1
AH016	200	PCR	2	8	2	FatP6.1
AH017	201	PCR	2	8	2	FatP6.1
AH018	202	PCR	2	8	2	FatP6.1
AH019	203	PCR	2	8	2	FatP6.1
AH020	204	PCR	2	8	2	FatP6.1
AH021	205	PCR	2	8	2	FatP6.1
AH022	206	PCR	2	8	2	FatP6.1
AH023	207	PCR	2	8	2	FatP6.1
AH024	208	PCR	2	8	2	FatP6.1
AH025	209	PCR	2	8	2	FatP6.1
AH026	210	PCR	2	8	2	FatP6.1
AH027	211	PCR	2	8	2	FatP6.1
AH028	212	PCR	2	8	2	FatP6.1
AH029	213	PCR	2	8	2	FatP6.1
AH030	214	PCR	2	8	2	FatP6.1
AH031	228	PCR	2	8	2	FatP6.1
AH032	229	PCR	2	8	2	FatP6.1
AH033	114	PCR	2	8	2	FatP6.1
AH034	117	PCR	2	8	2	FatP6.1
AH035	120	PCR	2	8	2	FatP6.1
AH036	123	PCR	2	8	2	FatP6.1
AH037	53	PCR	2	8	2	FatP6.1

20241120

These were dropped off for sequencing on 20241120 by Hollie.

Poc Histone RFLP

20241117

I performed an RFLP using the protocol linked above for all Pocillopora samples. I decided to run all samples before all have been sequenced because I will not have enough time to get back sequences and analyze before I leave URI. By performing RFLP on all samples, I will have the RFLP data to reference after analyzing sequences and will not require someone to run the RFLP for me later.

I ran the RFLP on 81 total samples.

I prepared the master mix.

Component	1 rxn	100 rxns
EmeraldAmp 2X TT master mix	12.55uL	1255ul
POC Histone F primer (10uM)	0.32uL	32uL
POC Histone R primer (10uM)	0.32uL	32uL
Ultrapure H20	10.8uL	1080uL
Template DNA	1uL
	25uL	23.99uL

I ran all reactions in a 96 well PCR plate. Added 24 uL of master mix to each well. Then added 1 uL of template DNA of each sample. 1uL of water was used in one well as a control.

I used a positive control as sample #159 from Hollie. There was very little DNA left in the tube.

The PCR protocol was as follows:

• 94°C for 60 sec [1 cycle]
• 94°C for 30 sec, 53°C for 30 sec, 72°C for 60 sec [30 cycles]
• 72°C for 5 min [1 cycle]
• Hold at 4°C

After PCR, samples were stored in the fridge overnight.

The plate map is included below. Note that the sample ID’s in this plate map are the well numbers of the gDNA from the original gDNA plate. The plate map for gDNA (below) will have the tube ID for this project.

20241118

After PCR for POC Histone, I ran the restriction enzyme incubation for the RFLP. I generated a master mix containing rCutSmart Buffer (1.9 uL per reaction) and XholRE (1.9 uL per reaction) with 15 uL of PCR product in each reaction.

I added the enzymes and sample to each well with 15 uL of water added to a new negative control (NEG2).

Samples were incubated at 37°C for 1 hr then at 65°C for 20 minutes with a 10°C hold in a thermocycler.

Samples were loaded in the same positions as the PCR plate. Sample ID in the plate maps indicates the well from the original gDNA plate.

I then looked at the PCR product on a gel after the enzyme incubation. If there is a cut, the sample is P. grandis and if the enzyme does not cut, the sample is P. meandrina. We are only going to use this to look at samples where the mtORF sequence analysis indicates that the sample is in haplotype 1a (grandis/meandrina complex) as described in linked protocols and papers above.

I ran 2% gels for 90 min at 80V to view the results. I ran this with 3 gels to fit all samples.

There are some samples that cut very clearly and negative controls have no bands. The positive control did not show up, which is not surprising because of the very low DNA available in the original tube. There are also some higher bands in some samples. The variation in banding pattern may not be surprising because we ran this on all samples, which may contain several Pocillopora species and not just P. meandrina and P. grandis, as it was designed for.

We will use these results to look for the cut bands for samples identified at haplotype 1a when sequence data is returned. The RFLP bands will not be used to identify species other than grandis/meandrina.

The enzyme did successfully cut and for samples we had preliminary sequences for, it did cut only those identified as haplotype 1a.

Porites

H2-H4 sequencing

20241117

Hollie prepared the Porites H2-H4 PCR for all Porites samples using the protocol linked above. The gDNA for these samples came from plates 40-41 of gDNA extractions.

We PCR’d samples from Plate 40 B6-H12 and 41 A1-A2 for 81 total samples.

Component	1 rxn	90 rxns
EmeraldAmp 2X TT master mix	12.6uL	1134ul
zH2AH4f F primer (10uM)	0.3uL	27uL
zH2FrR R primer (10uM)	0.3uL	27uL
Ultrapure H20	10.8uL	972uL
Template DNA	1uL
	25uL	23.99uL

The PCR protocol was as follows:

• 96°C for 120 sec [1 cycle]
• 96°C for 20 sec, 58.5°C for 20 sec, 72°C for 90 sec, 72°C for 90 sec [34 cycles]
• 72°C for 5 min [1 cycle]
• Hold at 4°C

We used a negative control of water only and used a positive control of a P. evermanni sample (JA 491 9/25/24).

Samples held overnight on the thermocycler at 4°C. We will run a gel in the morning.

The plate map sample ID refers to the tube ID, which is also what is recorded in the gDNA plate maps above.

20241118

I ran a gel to look at the PCR product. I ran a 1.5% gel at 80V for 90 min with 3uL of sample. I also added a 1Kb ladder. I ran three gels to fit all samples.

Notes from gel results:

• Wells A1, A2, A9-A12 had no bands
• All other samples have a clear band
• No bands in negative control
• Postive control has a clear band

I then looked at the gDNA data and some of the samples had >10 ng/uL DNA concentrations, which is about twice of that of samples that amplified well. I then decided to re-amplify the samples that failed with half of the template DNA, because the DNA concentration may have been too high for this PCR reaction.

I therefore did another round of PCR for the failed samples with 1/2 of the DNA volume (0.5 uL of DNA and 0.5 uL of water in each reaction).

Component	1 rxn	10 rxns
EmeraldAmp 2X TT master mix	12.55uL	125.54ul
zH2AH4f F primer (10uM)	0.32uL	3.2uL
zH2FrR R primer (10uM)	0.32uL	3.2uL
Ultrapure H20	11.3uL	113uL
Template DNA	0.5uL
	25uL	23.99uL

I used sample #75, which was a sample that successfully amplified yesterday as a postive control. Water was used as a negative control.

If these successfully amplify, they will replace the failed sample wells in the PCR product plate to submit for sequencing.

Ran in strip tubes labeled with tube ID. PCR ran as described above and I’ll run a gel in the morning.

20241119

I ran a 1.5% gel at 80V for 90min with 4uL of sample to check PCR product from samples that were re-done yesterday. There are 6 samples and 1 positive control, with 1 negative control.

All samples have bands - reducing DNA input worked! Positive control amplified and negative controls have no band.

We can now proceed with cleaning and preparation for sequencing for all Porites samples.

I then performed a clean up using the Zymo Zr-96 DNA clean up kit in a 96-well plate format. Note that during this process I removed the failed samples from the first round of PCR and replaced with the successful PCR product for those samples.

1. Add 100 uL DNA binding buffer to PCR samples, mix, and transfer to silicon A plate on top of column plate.
2. Centrifuge at 3000g for 5 min.
3. Add 300 uL of DNA wash buffer (100% ethanol added to buffer by HP) to each well.
4. Centrifugure at 3000g for 5 min.
5. Add 300 uL of DNA wash buffer again to each well.
6. Centrifugure at 3000g for 5 min.
7. Add 40uL nuclease free water to each well.
8. Transfer the silicon A plate to the elution plate.
9. Centrifuge at 3000g for 3 min.
10. DNA is now in the elution plate.
11. Store at -20°C until sending for sequencing.
12. Discard of waste in hood.

I then assembled boxes for each of the projects for sequencing. Project 1 is the Pocillopora mtORF samples as described above. Project 2 is the Porites H2 samples. To prepare for sequencing, I added the plate of clean DNA to a labeled box and added in a tube of 3.2 uM primers. We decided to sequence both the forward and reverse due to the length of the sequence desired.

Sample information is as follows for sequencing. Sample name is a running number for Janet and the Sample Name includes the actual tube ID that I will use to track to sample.

Forward primer:

Sample ID	Sample Name	Well	Template Type	PCR Template Volume (µl)	H20 Volume (µl)	Primer Volume (µl)	Primer Name	Notes
AH038	68	A1	PCR				zH2AH4f
AH039	124	B1	PCR				zH2AH4f
AH040	138	C1	PCR				zH2AH4f
AH041	81	D1	PCR				zH2AH4f
AH042	225	E1	PCR				zH2AH4f
AH043	232	F1	PCR				zH2AH4f
AH044	251	G1	PCR				zH2AH4f
EMPTY	NA	H1	PCR				zH2AH4f
AH045	69	A2	PCR				zH2AH4f
AH046	127	B2	PCR				zH2AH4f
AH047	140	C2	PCR				zH2AH4f
AH048	83	D2	PCR				zH2AH4f
AH049	226	E2	PCR				zH2AH4f
AH050	233	F2	PCR				zH2AH4f
AH051	192	G2	PCR				zH2AH4f
EMPTY	NA	H2	PCR				zH2AH4f
AH052	71	A3	PCR				zH2AH4f
AH053	128	B3	PCR				zH2AH4f
AH054	141	C3	PCR				zH2AH4f
AH055	86	D3	PCR				zH2AH4f
AH056	244	E3	PCR				zH2AH4f
AH057	234	F3	PCR				zH2AH4f
AH058	215	G3	PCR				zH2AH4f
EMPTY	NA	H3	PCR				zH2AH4f
AH059	75	A4	PCR				zH2AH4f
AH060	129	B4	PCR				zH2AH4f
AH061	65	C4	PCR				zH2AH4f
AH062	87	D4	PCR				zH2AH4f
AH063	245	E4	PCR				zH2AH4f
AH064	235	F4	PCR				zH2AH4f
AH065	217	G4	PCR				zH2AH4f
EMPTY	NA	H4	PCR				zH2AH4f
AH066	80	A5	PCR				zH2AH4f
AH067	130	B5	PCR				zH2AH4f
AH068	67	C5	PCR				zH2AH4f
AH069	90	D5	PCR				zH2AH4f
AH070	246	E5	PCR				zH2AH4f
AH071	238	F5	PCR				zH2AH4f
AH072	218	G5	PCR				zH2AH4f
EMPTY	NA	H5	PCR				zH2AH4f
AH073	82	A6	PCR				zH2AH4f
AH074	131	B6	PCR				zH2AH4f
AH075	70	C6	PCR				zH2AH4f
AH076	91	D6	PCR				zH2AH4f
AH077	247	E6	PCR				zH2AH4f
AH078	239	F6	PCR				zH2AH4f
AH079	220	G6	PCR				zH2AH4f
EMPTY	NA	H6	PCR				zH2AH4f
AH080	84	A7	PCR				zH2AH4f
AH081	132	B7	PCR				zH2AH4f
AH082	72	C7	PCR				zH2AH4f
AH083	92	D7	PCR				zH2AH4f
AH084	193	E7	PCR				zH2AH4f
AH085	240	F7	PCR				zH2AH4f
AH086	237	G7	PCR				zH2AH4f
EMPTY	NA	H7	PCR				zH2AH4f
AH087	85	A8	PCR				zH2AH4f
AH088	133	B8	PCR				zH2AH4f
AH089	73	C8	PCR				zH2AH4f
AH090	93	D8	PCR				zH2AH4f
AH091	216	E8	PCR				zH2AH4f
AH092	242	F8	PCR				zH2AH4f
AH093	241	G8	PCR				zH2AH4f
EMPTY	NA	H8	PCR				zH2AH4f
AH094	88	A9	PCR				zH2AH4f
AH095	134	B9	PCR				zH2AH4f
AH096	74	C9	PCR				zH2AH4f
AH097	221	D9	PCR				zH2AH4f
AH098	219	E9	PCR				zH2AH4f
AH099	243	F9	PCR				zH2AH4f
AH100	66	G9	PCR				zH2AH4f
EMPTY	NA	H9	PCR				zH2AH4f
AH101	94	A10	PCR				zH2AH4f
AH102	135	B10	PCR				zH2AH4f
AH103	76	C10	PCR				zH2AH4f
AH104	222	D10	PCR				zH2AH4f
AH105	227	E10	PCR				zH2AH4f
AH106	248	F10	PCR				zH2AH4f
AH107	Positive-JA491	G10	PCR				zH2AH4f
EMPTY	NA	H10	PCR				zH2AH4f
AH108	95	A11	PCR				zH2AH4f
AH109	136	B11	PCR				zH2AH4f
AH110	77	C11	PCR				zH2AH4f
AH111	223	D11	PCR				zH2AH4f
AH112	230	E11	PCR				zH2AH4f
AH113	249	F11	PCR				zH2AH4f
NA	negative-control	G11	PCR				zH2AH4f	PCR negative, do not sequence
EMPTY	NA	H11	PCR				zH2AH4f
AH115	96	A12	PCR				zH2AH4f
AH116	137	B12	PCR				zH2AH4f
AH117	79	C12	PCR				zH2AH4f
AH118	224	D12	PCR				zH2AH4f
AH119	231	E12	PCR				zH2AH4f
AH120	250	F12	PCR				zH2AH4f
NA	negative-control	G12	PCR				zH2AH4f	PCR negative, do not sequence
EMPTY	NA	H12	PCR				zH2AH4f

Reverse primer:

Sample ID	Sample Name	Well	Template Type	PCR Template Volume (µl)	H20 Volume (µl)	Primer Volume (µl)	Primer Name	Notes
AH121	68	A1	PCR				zH4Fr
AH122	124	B1	PCR				zH4Fr
AH123	138	C1	PCR				zH4Fr
AH124	81	D1	PCR				zH4Fr
AH125	225	E1	PCR				zH4Fr
AH126	232	F1	PCR				zH4Fr
AH127	251	G1	PCR				zH4Fr
EMPTY	NA	H1	PCR				zH4Fr
AH128	69	A2	PCR				zH4Fr
AH129	127	B2	PCR				zH4Fr
AH130	140	C2	PCR				zH4Fr
AH131	83	D2	PCR				zH4Fr
AH132	226	E2	PCR				zH4Fr
AH133	233	F2	PCR				zH4Fr
AH134	192	G2	PCR				zH4Fr
EMPTY	NA	H2	PCR				zH4Fr
AH135	71	A3	PCR				zH4Fr
AH136	128	B3	PCR				zH4Fr
AH137	141	C3	PCR				zH4Fr
AH138	86	D3	PCR				zH4Fr
AH139	244	E3	PCR				zH4Fr
AH140	234	F3	PCR				zH4Fr
AH141	215	G3	PCR				zH4Fr
EMPTY	NA	H3	PCR				zH4Fr
AH142	75	A4	PCR				zH4Fr
AH143	129	B4	PCR				zH4Fr
AH144	65	C4	PCR				zH4Fr
AH145	87	D4	PCR				zH4Fr
AH146	245	E4	PCR				zH4Fr
AH147	235	F4	PCR				zH4Fr
AH148	217	G4	PCR				zH4Fr
EMPTY	NA	H4	PCR				zH4Fr
AH149	80	A5	PCR				zH4Fr
AH150	130	B5	PCR				zH4Fr
AH151	67	C5	PCR				zH4Fr
AH152	90	D5	PCR				zH4Fr
AH153	246	E5	PCR				zH4Fr
AH154	238	F5	PCR				zH4Fr
AH155	218	G5	PCR				zH4Fr
EMPTY	NA	H5	PCR				zH4Fr
AH156	82	A6	PCR				zH4Fr
AH157	131	B6	PCR				zH4Fr
AH158	70	C6	PCR				zH4Fr
AH159	91	D6	PCR				zH4Fr
AH160	247	E6	PCR				zH4Fr
AH161	239	F6	PCR				zH4Fr
AH162	220	G6	PCR				zH4Fr
EMPTY	NA	H6	PCR				zH4Fr
AH162	84	A7	PCR				zH4Fr
AH163	132	B7	PCR				zH4Fr
AH164	72	C7	PCR				zH4Fr
AH165	92	D7	PCR				zH4Fr
AH166	193	E7	PCR				zH4Fr
AH167	240	F7	PCR				zH4Fr
AH168	237	G7	PCR				zH4Fr
EMPTY	NA	H7	PCR				zH4Fr
AH169	85	A8	PCR				zH4Fr
AH170	133	B8	PCR				zH4Fr
AH171	73	C8	PCR				zH4Fr
AH172	93	D8	PCR				zH4Fr
AH173	216	E8	PCR				zH4Fr
AH174	242	F8	PCR				zH4Fr
AH175	241	G8	PCR				zH4Fr
EMPTY	NA	H8	PCR				zH4Fr
AH176	88	A9	PCR				zH4Fr
AH177	134	B9	PCR				zH4Fr
AH178	74	C9	PCR				zH4Fr
AH179	221	D9	PCR				zH4Fr
AH180	219	E9	PCR				zH4Fr
AH181	243	F9	PCR				zH4Fr
AH182	66	G9	PCR				zH4Fr
EMPTY	NA	H9	PCR				zH4Fr
AH183	94	A10	PCR				zH4Fr
AH184	135	B10	PCR				zH4Fr
AH185	76	C10	PCR				zH4Fr
AH186	222	D10	PCR				zH4Fr
AH187	227	E10	PCR				zH4Fr
AH188	248	F10	PCR				zH4Fr
AH189	Positive-JA491	G10	PCR				zH4Fr
EMPTY	NA	H10	PCR				zH4Fr
AH190	95	A11	PCR				zH4Fr
AH191	136	B11	PCR				zH4Fr
AH192	77	C11	PCR				zH4Fr
AH193	223	D11	PCR				zH4Fr
AH194	230	E11	PCR				zH4Fr
AH195	249	F11	PCR				zH4Fr
NA	negative-control	G11	PCR				zH4Fr	PCR negative, do not sequence
EMPTY	NA	H11	PCR				zH4Fr
AH196	96	A12	PCR				zH4Fr
AH197	137	B12	PCR				zH4Fr
AH198	79	C12	PCR				zH4Fr
AH199	224	D12	PCR				zH4Fr
AH200	231	E12	PCR				zH4Fr
AH201	250	F12	PCR				zH4Fr
NA	negative-control	G12	PCR				zH4Fr	PCR negative, do not sequence
EMPTY	NA	H12	PCR				zH4Fr

20241120

Samples were dropped off by Hollie today for sequencing.

Acropora

Primer selection

20241119

Hollie and I talked about primers to use for Acropora species identification. Hollie previously tested some of the primers from the literature. We already have primers in the lab for PaxC (PaxC_intron_FP1 forward and PaxC_intron_RP1 reverse) and CRf/CO3r (CRF forward and CO3r reverse) so we will try those first.

PaxC_intron:

• van Oppen et al. 2004 and van Oppen et al. 2001

CRf/CO3r:

• Mitochondrial putative control (933+ bp) + 83 bp of cytochrome oxidase III as in Vollmer et al. 2002

CRf/CO3r sequencing and PaxC_intron_FP1/PaxC_intron_RP1 sequencing

I prepared two PCR plates - one for each primer set.

CRf/CO3r:

Component	1 rxn	95 rxns
EmeraldAmp 2X TT master mix	12.55uL	1192.25ul
Ultrapure H20	10.80uL	1026uL
CRf primer (10uM)	0.32uL	30.4uL
CO3r primer (10uM)	0.32uL	30.4uL
Template DNA	1uL
	25uL	23.99uL

PaxC:

Component	1 rxn	95 rxns
EmeraldAmp 2X TT master mix	12.55uL	1192.25ul
Ultrapure H20	10.80uL	1026uL
PaxC FP1 primer (10uM)	0.32uL	30.4uL
PaxC RP1 primer (10uM)	0.32uL	30.4uL
Template DNA	1uL
	25uL	23.99uL

I used a Becker Heatwave Acropora pulchra as a postive control (no. 24 gDNA from 10/29/23). Water was used as a negative control in both plates.

The plate maps for these PCRs shows the sample ID as the well from the original gDNA plates.

The gDNA plates are as follows.

The PCR protocol (“acro” on Putnam account) was the same for both primers.

• 95°C for 3 min [1 cycle]
• 94°C for 30 sec, 53°C for 30 sec, 72°C for 1 min [35 cycles] melting temperature chosen based on lowest primer melting temperature
• 72°C for 5 min
• Hold at 4°C

Images of primer labels can be found on GitHub here.

I then ran a gel for the CRf/CO3r primer set. I’ll run the PaxC gel in the morning.

I ran PCR product on a 2% gel for 90 min at 80V.

The gel product looks really nice. No bands in the negative controls, a band in the positive control. There are bands present for all other samples. Sample AH229 had a faint band - but this is ok because it was an accidental Pocillopora sample that was included! All other samples look good.

20241120

I ran a gel for the PaxC PCR products using a 2% gel at 80V for 90 min.

Notes from the gel results:

• Very interesting! The bands all have varying lengths. It will be interesting to see if band length corresponds with species after sequencing analyses.
• Negative controls have no bad, the positive control as a band.
• Interestingly, the Pocillopora sample amplified well (we won’t be analyzing this sample since it was an accidental inclusion).

All samples look good, we will move forward with cleaning and sample preps.

I cleaned the plates following the same protocol as above for Porites samples. Sample plates were labeled and stored in the freezer with one plate for each primer set.

We will sequence both the forward and reverse for the CRf/CO3r primer set and only the forward for the PaxC (smaller length).

Sample info is as follows.

CRf/CO3r forward:

Sample ID	Sample Name	Well	Template Type	PCR Template Volume (µl)	H20 Volume (µl)	Primer Volume (µl)	Primer Name
AH202	13	A1	PCR				CRf
AH203	102	B1	PCR				CRf
AH204	4	C1	PCR				CRf
AH205	26	D1	PCR				CRf
AH206	151	E1	PCR				CRf
AH207	163	F1	PCR				CRf
AH208	171	G1	PCR				CRf
EMPTY	NA	H1	PCR				CRf
AH209	14	A2	PCR				CRf
AH210	103	B2	PCR				CRf
AH211	6	C2	PCR				CRf
AH212	27	D2	PCR				CRf
AH213	152	E2	PCR				CRf
AH214	164	F2	PCR				CRf
AH215	172	G2	PCR				CRf
EMPTY	NA	H2	PCR				CRf
AH216	15	A3	PCR				CRf
AH217	104	B3	PCR				CRf
AH218	7	C3	PCR				CRf
AH219	30	D3	PCR				CRf
AH220	153	E3	PCR				CRf
AH221	166	F3	PCR				CRf
AH222	174	G3	PCR				CRf
EMPTY	NA	H3	PCR				CRf
AH223	16	A4	PCR				CRf
AH224	105	B4	PCR				CRf
AH225	8	C4	PCR				CRf
AH226	31	D4	PCR				CRf
AH227	154	E4	PCR				CRf
AH228	168	F4	PCR				CRf
AH229	175	G4	PCR				CRf
EMPTY	NA	H4	PCR				CRf
AH230	20	A5	PCR				CRf
AH231	106	B5	PCR				CRf
AH232	9	C5	PCR				CRf
AH233	32	D5	PCR				CRf
AH234	155	E5	PCR				CRf
AH235	173	F5	PCR				CRf
AH236	177	G5	PCR				CRf
EMPTY	NA	H5	PCR				CRf
AH237	28	A6	PCR				CRf
AH238	107	B6	PCR				CRf
AH239	12	C6	PCR				CRf
AH240	142	D6	PCR				CRf
AH241	156	E6	PCR				CRf
AH242	176	F6	PCR				CRf
AH243	229	G6	PCR				CRf
EMPTY	NA	H6	PCR				CRf
AH244	29	A7	PCR				CRf
AH245	108	B7	PCR				CRf
AH246	17	C7	PCR				CRf
AH247	143	D7	PCR				CRf
AH248	157	E7	PCR				CRf
AH249	178	F7	PCR				CRf
AH250	1	G7	PCR				CRf
EMPTY	NA	H7	PCR				CRf
AH251	97	A8	PCR				CRf
AH252	109	B8	PCR				CRf
AH253	18	C8	PCR				CRf
AH254	144	D8	PCR				CRf
AH255	158	E8	PCR				CRf
AH256	148	F8	PCR				CRf
AH257	5	G8	PCR				CRf
EMPTY	NA	H8	PCR				CRf
AH258	98	A9	PCR				CRf
AH259	110	B9	PCR				CRf
AH260	19	C9	PCR				CRf
AH261	145	D9	PCR				CRf
AH262	159	E9	PCR				CRf
AH263	150	F9	PCR				CRf
AH264	10	G9	PCR				CRf
EMPTY	NA	H9	PCR				CRf
AH265	99	A10	PCR				CRf
AH266	111	B10	PCR				CRf
AH267	21	C10	PCR				CRf
AH268	146	D10	PCR				CRf
AH269	160	E10	PCR				CRf
AH270	165	F10	PCR				CRf
AH271	11	G10	PCR				CRf
EMPTY	NA	H10	PCR				CRf
AH272	100	A11	PCR				CRf
AH273	2	B11	PCR				CRf
AH274	24	C11	PCR				CRf
AH275	147	D11	PCR				CRf
AH276	161	E11	PCR				CRf
AH277	167	F11	PCR				CRf
NA	NA	G11	PCR				CRf
EMPTY	NA	H11	PCR				CRf
AH278	101	A12	PCR				CRf
AH279	3	B12	PCR				CRf
AH280	25	C12	PCR				CRf
AH281	149	D12	PCR				CRf
AH282	162	E12	PCR				CRf
AH283	170	F12	PCR				CRf
AH284	Becker24	G12	PCR				CRf
EMPTY	NA	H12	PCR				CRf

CRf/CO3r reverse:

Sample ID	Sample Name	Well	Template Type	PCR Template Volume (µl)	H20 Volume (µl)	Primer Volume (µl)	Primer Name
AH285	13	A1	PCR				CO3r
AH286	102	B1	PCR				CO3r
AH287	4	C1	PCR				CO3r
AH288	26	D1	PCR				CO3r
AH289	151	E1	PCR				CO3r
AH290	163	F1	PCR				CO3r
AH291	171	G1	PCR				CO3r
EMPTY	NA	H1	PCR				CO3r
AH292	14	A2	PCR				CO3r
AH293	103	B2	PCR				CO3r
AH294	6	C2	PCR				CO3r
AH295	27	D2	PCR				CO3r
AH296	152	E2	PCR				CO3r
AH297	164	F2	PCR				CO3r
AH298	172	G2	PCR				CO3r
EMPTY	NA	H2	PCR				CO3r
AH299	15	A3	PCR				CO3r
AH300	104	B3	PCR				CO3r
AH301	7	C3	PCR				CO3r
AH302	30	D3	PCR				CO3r
AH303	153	E3	PCR				CO3r
AH304	166	F3	PCR				CO3r
AH305	174	G3	PCR				CO3r
EMPTY	NA	H3	PCR				CO3r
AH306	16	A4	PCR				CO3r
AH307	105	B4	PCR				CO3r
AH308	8	C4	PCR				CO3r
AH309	31	D4	PCR				CO3r
AH310	154	E4	PCR				CO3r
AH311	168	F4	PCR				CO3r
AH312	175	G4	PCR				CO3r
EMPTY	NA	H4	PCR				CO3r
AH313	20	A5	PCR				CO3r
AH314	106	B5	PCR				CO3r
AH315	9	C5	PCR				CO3r
AH316	32	D5	PCR				CO3r
AH317	155	E5	PCR				CO3r
AH318	173	F5	PCR				CO3r
AH319	177	G5	PCR				CO3r
EMPTY	NA	H5	PCR				CO3r
AH320	28	A6	PCR				CO3r
AH321	107	B6	PCR				CO3r
AH322	12	C6	PCR				CO3r
AH323	142	D6	PCR				CO3r
AH324	156	E6	PCR				CO3r
AH325	176	F6	PCR				CO3r
AH326	229	G6	PCR				CO3r
EMPTY	NA	H6	PCR				CO3r
AH327	29	A7	PCR				CO3r
AH328	108	B7	PCR				CO3r
AH329	17	C7	PCR				CO3r
AH330	143	D7	PCR				CO3r
AH331	157	E7	PCR				CO3r
AH332	178	F7	PCR				CO3r
AH333	1	G7	PCR				CO3r
EMPTY	NA	H7	PCR				CO3r
AH334	97	A8	PCR				CO3r
AH335	109	B8	PCR				CO3r
AH336	18	C8	PCR				CO3r
AH337	144	D8	PCR				CO3r
AH338	158	E8	PCR				CO3r
AH339	148	F8	PCR				CO3r
AH340	5	G8	PCR				CO3r
EMPTY	NA	H8	PCR				CO3r
AH341	98	A9	PCR				CO3r
AH342	110	B9	PCR				CO3r
AH343	19	C9	PCR				CO3r
AH344	145	D9	PCR				CO3r
AH345	159	E9	PCR				CO3r
AH346	150	F9	PCR				CO3r
AH347	10	G9	PCR				CO3r
EMPTY	NA	H9	PCR				CO3r
AH348	99	A10	PCR				CO3r
AH349	111	B10	PCR				CO3r
AH350	21	C10	PCR				CO3r
AH351	146	D10	PCR				CO3r
AH352	160	E10	PCR				CO3r
AH353	165	F10	PCR				CO3r
AH354	11	G10	PCR				CO3r
EMPTY	NA	H10	PCR				CO3r
AH355	100	A11	PCR				CO3r
AH356	2	B11	PCR				CO3r
AH357	24	C11	PCR				CO3r
AH358	147	D11	PCR				CO3r
AH359	161	E11	PCR				CO3r
AH360	167	F11	PCR				CO3r
NA	NA	G11	PCR				CO3r
EMPTY	NA	H11	PCR				CO3r
AH361	101	A12	PCR				CO3r
AH362	3	B12	PCR				CO3r
AH363	25	C12	PCR				CO3r
AH364	149	D12	PCR				CO3r
AH365	162	E12	PCR				CO3r
AH366	170	F12	PCR				CO3r
AH367	Becker24	G12	PCR				CO3r
EMPTY	NA	H12	PCR				CO3r

PaxC forward:

Sample ID	Sample Name	Well	Template Type	PCR Template Volume (µl)	H20 Volume (µl)	Primer Volume (µl)	Primer Name
AH368	13	A1	PCR				PaxC_intron_FP1
AH369	102	B1	PCR				PaxC_intron_FP1
AH370	4	C1	PCR				PaxC_intron_FP1
AH371	26	D1	PCR				PaxC_intron_FP1
AH372	151	E1	PCR				PaxC_intron_FP1
AH373	163	F1	PCR				PaxC_intron_FP1
AH374	171	G1	PCR				PaxC_intron_FP1
EMPTY	NA	H1	PCR				PaxC_intron_FP1
AH375	14	A2	PCR				PaxC_intron_FP1
AH376	103	B2	PCR				PaxC_intron_FP1
AH377	6	C2	PCR				PaxC_intron_FP1
AH378	27	D2	PCR				PaxC_intron_FP1
AH379	152	E2	PCR				PaxC_intron_FP1
AH380	164	F2	PCR				PaxC_intron_FP1
AH381	172	G2	PCR				PaxC_intron_FP1
EMPTY	NA	H2	PCR				PaxC_intron_FP1
AH382	15	A3	PCR				PaxC_intron_FP1
AH383	104	B3	PCR				PaxC_intron_FP1
AH384	7	C3	PCR				PaxC_intron_FP1
AH385	30	D3	PCR				PaxC_intron_FP1
AH386	153	E3	PCR				PaxC_intron_FP1
AH387	166	F3	PCR				PaxC_intron_FP1
AH388	174	G3	PCR				PaxC_intron_FP1
EMPTY	NA	H3	PCR				PaxC_intron_FP1
AH389	16	A4	PCR				PaxC_intron_FP1
AH390	105	B4	PCR				PaxC_intron_FP1
AH391	8	C4	PCR				PaxC_intron_FP1
AH392	31	D4	PCR				PaxC_intron_FP1
AH393	154	E4	PCR				PaxC_intron_FP1
AH394	168	F4	PCR				PaxC_intron_FP1
AH395	175	G4	PCR				PaxC_intron_FP1
EMPTY	NA	H4	PCR				PaxC_intron_FP1
AH396	20	A5	PCR				PaxC_intron_FP1
AH397	106	B5	PCR				PaxC_intron_FP1
AH398	9	C5	PCR				PaxC_intron_FP1
AH399	32	D5	PCR				PaxC_intron_FP1
AH400	155	E5	PCR				PaxC_intron_FP1
AH401	173	F5	PCR				PaxC_intron_FP1
AH402	177	G5	PCR				PaxC_intron_FP1
EMPTY	NA	H5	PCR				PaxC_intron_FP1
AH403	28	A6	PCR				PaxC_intron_FP1
AH404	107	B6	PCR				PaxC_intron_FP1
AH405	12	C6	PCR				PaxC_intron_FP1
AH406	142	D6	PCR				PaxC_intron_FP1
AH407	156	E6	PCR				PaxC_intron_FP1
AH408	176	F6	PCR				PaxC_intron_FP1
AH409	11	G6	PCR				PaxC_intron_FP1
EMPTY	NA	H6	PCR				PaxC_intron_FP1
AH410	29	A7	PCR				PaxC_intron_FP1
AH411	108	B7	PCR				PaxC_intron_FP1
AH412	17	C7	PCR				PaxC_intron_FP1
AH413	143	D7	PCR				PaxC_intron_FP1
AH414	157	E7	PCR				PaxC_intron_FP1
AH415	178	F7	PCR				PaxC_intron_FP1
AH416	229	G7	PCR				PaxC_intron_FP1
EMPTY	NA	H7	PCR				PaxC_intron_FP1
AH417	97	A8	PCR				PaxC_intron_FP1
AH418	109	B8	PCR				PaxC_intron_FP1
AH419	18	C8	PCR				PaxC_intron_FP1
AH420	144	D8	PCR				PaxC_intron_FP1
AH421	158	E8	PCR				PaxC_intron_FP1
AH422	148	F8	PCR				PaxC_intron_FP1
AH423	1	G8	PCR				PaxC_intron_FP1
EMPTY	NA	H8	PCR				PaxC_intron_FP1
AH424	98	A9	PCR				PaxC_intron_FP1
AH425	110	B9	PCR				PaxC_intron_FP1
AH426	19	C9	PCR				PaxC_intron_FP1
AH427	145	D9	PCR				PaxC_intron_FP1
AH428	159	E9	PCR				PaxC_intron_FP1
AH429	150	F9	PCR				PaxC_intron_FP1
AH430	5	G9	PCR				PaxC_intron_FP1
EMPTY	NA	H9	PCR				PaxC_intron_FP1
AH431	99	A10	PCR				PaxC_intron_FP1
AH432	111	B10	PCR				PaxC_intron_FP1
AH433	21	C10	PCR				PaxC_intron_FP1
AH434	146	D10	PCR				PaxC_intron_FP1
AH435	160	E10	PCR				PaxC_intron_FP1
AH436	165	F10	PCR				PaxC_intron_FP1
AH437	10	G10	PCR				PaxC_intron_FP1
EMPTY	NA	H10	PCR				PaxC_intron_FP1
AH438	100	A11	PCR				PaxC_intron_FP1
AH439	2	B11	PCR				PaxC_intron_FP1
AH440	24	C11	PCR				PaxC_intron_FP1
AH441	147	D11	PCR				PaxC_intron_FP1
AH442	161	E11	PCR				PaxC_intron_FP1
AH443	167	F11	PCR				PaxC_intron_FP1
NA	NA	G11	PCR				PaxC_intron_FP1
EMPTY	NA	H11	PCR				PaxC_intron_FP1
AH444	101	A12	PCR				PaxC_intron_FP1
AH445	3	B12	PCR				PaxC_intron_FP1
AH446	25	C12	PCR				PaxC_intron_FP1
AH447	149	D12	PCR				PaxC_intron_FP1
AH448	162	E12	PCR				PaxC_intron_FP1
AH449	170	F12	PCR				PaxC_intron_FP1
AH450	Becker24	G12	PCR				PaxC_intron_FP1
EMPTY	NA	H12	PCR				PaxC_intron_FP1

20241120

Hollie dropped these off for sequencing.

Next steps

Next, I will analyze sequences to determine species ID and then decide how to proceed with sample processing for RNA and/or metabolomics.