Heron Island Pdam Species ID (mtORF confirmation) of samples that were originally not able to be amplified via PCR and Sanger Sequencing

See base protocol and notes from Hollie

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

Sample List: RF16, RF24, RS02, RS11

colony_id	Extraction Date	Tech	Primerset	PCR Date
RF16A	20221111	ZD	mtORF	4/12/2023
RF24B	20221102	ZD	mtORF	4/12/2023
RS02C	20221118	ZD	mtORF	4/11/2023
RS11D	20221111	ZD	mtORF	4/11/2023

Amplification of 4 POC samples with mtORF on 4/11/2023.

Positive control: Kristina’s E5 extracted DNA Tube #383 (POC 45).

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

(cropped from large gel with E5 samples, for original gel image see: (https://github.com/zdellaert/ZD_Putnam_Lab_Notebook/blob/master/images/gels/originals/2023-04-11-gel-orig.JPG)

Had to redo RF16 and RF24 on 4/12/23

Qubit Values:

colony_id	Qubit 1	Qubit 2	Qubit AVG	Nanodrop
RF16A	13.40	13.40	13.40	113.30
RF24B	15.70	15.70	15.70	109.90
RS02C	6.82	7.28	7.05	90.10
RS11D	9.22	9.54	9.38	88.60

Heron Island Pdam POC mtORF cleaned PCR products (ZD61-68) will be sent for sequencing on 4/18/2023.

SET UP USING QUBIT VALUES, so did not dilute the PCR product.

colony_id	Extraction Date	Tech	Primerset	PCR Date	PCR Notes	GSC Sanger Date	GSC Sample #1	GSC Sample #2
RF16A	20221111	ZD	mtORF	4/12/2023		4/18/2023	ZD61_230418	ZD62_230418
RF24B	20221102	ZD	mtORF	4/12/2023		4/18/2023	ZD63_230418	ZD64_230418
RS02C	20221118	ZD	mtORF	4/11/2023		4/18/2023	ZD65_230418	ZD66_230418
RS11D	20221111	ZD	mtORF	4/11/2023		4/18/2023	ZD67_230418	ZD68_230418

Sample ID	Well	Template	A. Template size	B. Template stock conc. (ng/ul)	C. PCR template: ng needed	D. Volume=(C/B)ul	E	F. Volume PCR H20 needed (10-D or E)	G. Primer Vol	Colony
61		PCR	1000	7.05	25	3.5		6.5	2	RS02C
62		PCR	1000	7.05	25	3.5		6.5	2	RS02C
63		PCR	1000	9.38	25	2.7		7.3	2	RS11D
64		PCR	1000	9.38	25	2.7		7.3	2	RS11D
65		PCR	1000	13.4	25	1.9		8.1	2	RF16A
66		PCR	1000	13.4	25	1.9		8.1	2	RF16A
67		PCR	1000	15.7	25	1.6		8.4	2	RF24B
68		PCR	1000	15.7	25	1.6		8.4	2	RF24B