E5 Species ID (POC and POR) via PCR and Sanger Sequencing

Link with sample list and metadata

See base protocol and notes from Hollie

Followed my PCR Protocol exactly, based on instruction from Kristina Terpis

Sample List: All POC and POR colonies from the E5 timeseries need to be ID’d at one of the timepoints. Starting with Site 1.

Amplification of 15 POC samples with mtORF on 3/14/2023.

n.	timepoint	collection_site	colony_id	Extraction Date	Tech	Primerset	PCR Date
383	TP2	site1	POC-45	20211001	KXT	mtORF	3/14/2023
385	TP2	site1	POC-47	20211012	KXT	mtORF	3/14/2023
387	TP2	site1	POC-44	20211105	KXT	mtORF	3/14/2023
389	TP2	site1	POC-42	20211116	KXT	mtORF	3/14/2023
391	TP2	site1	POC-55	20211118	KXT	mtORF	3/14/2023
393	TP2	site1	POC-50	20210903	KXT	mtORF	3/14/2023
395	TP2	site1	POC-56	20211028	KXT	mtORF	3/14/2023
397	TP2	site1	POC-40	20210927	KXT	mtORF	3/14/2023
399	TP2	site1	POC-52	20211102	KXT	mtORF	3/14/2023
401	TP2	site1	POC-53	20211118	KXT	mtORF	3/14/2023
403	TP2	site1	POC-48	20211129	KXT	mtORF	3/14/2023
405	TP2	site1	POC-68	20211001	KXT	mtORF	3/14/2023
407	TP2	site1	POC-41	20211015	KXT	mtORF	3/14/2023
411	TP2	site1	POC-43	20210902	KXT	mtORF	3/14/2023
417	TP2	site1	POC-57	20211020	KXT	mtORF	3/14/2023

Positive control: Kristina’s POC sample from Pierrick’s sample #203 (diluted 1:10).

DNA and RNA Quality Check: 1% Gel at 80V for 30 mins.

Nanodrop of cleaned PCR product

Amplification of 11 the above POC samples with PocHistone on 3/27/2023.

n.	timepoint	collection_site	colony_id	Extraction Date	Tech	Primerset	PCR Date
385	TP2	site1	POC-47	20211012	KXT	PocHistone	3/27/2023
387	TP2	site1	POC-44	20211105	KXT	PocHistone	3/27/2023
389	TP2	site1	POC-42	20211116	KXT	PocHistone	3/27/2023
393	TP2	site1	POC-50	20210903	KXT	PocHistone	3/27/2023
395	TP2	site1	POC-56	20211028	KXT	PocHistone	3/27/2023
399	TP2	site1	POC-52	20211102	KXT	PocHistone	3/27/2023
401	TP2	site1	POC-53	20211118	KXT	PocHistone	3/27/2023
403	TP2	site1	POC-48	20211129	KXT	PocHistone	3/27/2023
405	TP2	site1	POC-68	20211001	KXT	PocHistone	3/27/2023
411	TP2	site1	POC-43	20210902	KXT	PocHistone	3/27/2023
417	TP2	site1	POC-57	20211020	KXT	PocHistone	3/27/2023

No postive control.

DNA and RNA Quality Check: 1% Gel at 80V for 30 mins.

Nanodrop of cleaned PCR product

Amplification of 15 POR samples on 3/15/2023.

n.	timepoint	collection_site	colony_id	Extraction Date	Tech
415	TP2	site1	POR-75	20210902	KXT
421	TP2	site1	POR-82	20211008	KXT
425	TP2	site1	POR-80	20211014	KXT
469	TP2	site1	POR-83	20220208	KXT
471	TP2	site1	POR-76	20211015	KXT
473	TP2	site1	POR-77	20211116	KXT
475	TP2	site1	POR-78	20210921	KXT
477	TP2	site1	POR-69	20211015	KXT
479	TP2	site1	POR-74	20211020	KXT
483	TP2	site1	POR-81	20220208	KXT
485	TP2	site1	POR-70	20211109	KXT
487	TP2	site1	POR-71	20211122	KXT
489	TP2	site1	POR-79	20211129	KXT
491	TP2	site1	POR-73	20211101	KXT
493	TP2	site1	POR-72	20211109	KXT

Positive control: Kristina’s E5 extracted DNA Tube #5 (extraction date: 20210826).

DNA and RNA Quality Check: 1% Gel at 80V for 30 mins.

Nanodrop of cleaned PCR product

POC mtORF (ZD01-30) and POR H2 (ZD33-62) were sent For Sanger Sequencing on 3/16/2023. POC PocHistone (ZD63-84) were sent for sequencing on 3/28/2023.