PCR 1 amplifying ITS2 marker for Box 3 - O.Fra deep

Protocol

Samples:

• OFAV = Orbicella faveolata
• OFRA = Orbicella franksi
• S = Shallow
• P = Deep

Species	Treatment	ID
OFRA	P	0CX4
OFRA	P	0CX5
OFRA	P	0GT1
OFRA	P	0GT3
OFRA	P	0GT5
OFRA	P	1GT36
OFRA	P	1GT37
OFRA	P	1GT42
OFRA	P	1GT43
OFRA	P	2CX10
OFRA	P	2CX12
OFRA	P	2CX13
OFRA	P	2CX8
OFRA	P	2GT10
OFRA	P	2GT3
OFRA	P	2GT31
OFRA	P	2GT38
OFRA	P	2GT41
OFRA	P	2GT7
OFRA	P	2HS11
OFRA	P	2HS17
OFRA	P	2HS27
OFRA	P	2HS9
OFRA	P	4CX12
OFRA	P	4HS13
OFRA	P	4HS15
OFRA	P	6CX18
OFRA	P	6EV18
OFRA	P	6GT17
OFRA	P	8CX24
OFRA	P	8HS21
OFRA	P	8HS23
OFRA	P	8HS24
OFRA	P	9CX5
OFRA	P	9CX6
OFRA	P	9GT30
OFRA	P	9GT47

Master Mix:

Component	Volume for 1 rxn	Volume for 37 rxns (+1% error)
2x MyTaq Mix	25.0 uL	1017.5 uL
Molecular Water	18.0 uL	732.6 uL
ITS2 Primer Forward (10 uM)	2.0 uL	81.4 uL
ITS2 Primer Reverse (10 uM)	2.0 uL	81.4 uL
DNA	3.0 uL	NA

Strip Tube Set Up:

Strip (replicate) Number	A	B	C	D	E	F	G	H
1	0CX4	0CX5	0GT1	0GT3	0GT5	1GT36	1GT37	1GT42
2	0CX4	0CX5	0GT1	0GT3	0GT5	1GT36	1GT37	1GT42
3	0CX4	0CX5	0GT1	0GT3	0GT5	1GT36	1GT37	1GT42
4	1GT43	2CX10	2CX12	2CX13	2CX8	2GT10	2GT3	2GT38
4	1GT43	2CX10	2CX12	2CX13	2CX8	2GT10	2GT3	2GT38
4	1GT43	2CX10	2CX12	2CX13	2CX8	2GT10	2GT3	2GT38
7	2GT41	2GT7	2HS11	2HS17	2HS27	2HS9	4CX12	4HS13
7	2GT41	2GT7	2HS11	2HS17	2HS27	2HS9	4CX12	4HS13
7	2GT41	2GT7	2HS11	2HS17	2HS27	2HS9	4CX12	4HS13
10	4HS15	6CX18	6EV18	6GT17	8CX24	8HS21	8HS23	8HS24
10	4HS15	6CX18	6EV18	6GT17	8CX24	8HS21	8HS23	8HS24
10	4HS15	6CX18	6EV18	6GT17	8CX24	8HS21	8HS23	8HS24
13	9CX5	9CX6	9GT30	9GT47
13	9CX5	9CX6	9GT30	9GT47
13	9CX5	9CX6	9GT30	9GT47

Gel Map:

Failed: 1GT42, 2CX13, 2GT3, 2HSQQ7, 2HS27, 6GT17, 8HS23, 9CX6, 9GT30

Notes: Large gel box electrical connection broke, but I realized after the gel had already been loaded. I cut the large gel and ran it in two medium gel boxes, however some wells may have failed due to cutting the gel and movement of gel from box to box