PCR 1 amplifying ITS2 marker for Box 4 - O.Fav shallow

Protocol

Samples:

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

Species	Treatment	ID
OFAV	S	0AZ4
OFAV	S	0DW4
OFAV	S	0EV1
OFAV	S	0EV3
OFAV	S	1AZ5
OFAV	S	2AZ2
OFAV	S	2AZ21
OFAV	S	BLANK
OFAV	S	2AZ7
OFAV	S	2DW40
OFAV	S	2DW44
OFAV	S	2EV10
OFAV	S	2EV3
OFAV	S	2EV30
OFAV	S	2EV34
OFAV	S	2EV36
OFAV	S	2EV44
OFAV	S	BLANK
OFAV	S	BLANK
OFAV	S	2JQ6
OFAV	S	2JQ8
OFAV	S	3AZ4
OFAV	S	3EV30
OFAV	S	3JQ1
OFAV	S	4DW11
OFAV	S	4DW14
OFAV	S	4EV14
OFAV	S	BLANK
OFAV	S	4JQ12
OFAV	S	6JQ16
OFAV	S	8EV24
OFAV	S	9AZ10
OFAV	S	9AZ7
OFAV	S	9AZ81
OFAV	S	9DW32
OFAV	S	9DW37
OFAV	S	9EV2
OFAV	S	9EVE41
OFAV	S	9JQ5
OFAV	S	JQ1

Samples from Box1 re diluted | OFAV | P | 9GT46 | | OFAV | P | 352 | | OFAV | P | 356 | | OFAV | P | 0HS3 | | OFAV | P | 9AZ5 |

Master Mix:

Component	Volume for 1 rxn	Volume for 45 rxns (+1% error)
2x MyTaq Mix	25.0 uL	1136.3 uL
Molecular Water	18.0 uL	818.1 uL
ITS2 Primer Forward (10 uM)	2.0 uL	90.9 uL
ITS2 Primer Reverse (10 uM)	2.0 uL	90.9 uL
DNA	3.0 uL	NA

Strip Tube Set Up:

Strip (replicate) Number	A	B	C	D	E	F	G	H
1	0AZ4	0DW46	0EV1	0EV3	1AZ5	2AZ2	2AZ21	B;ANK
1	0AZ4	0DW46	0EV1	0EV3	1AZ5	2AZ2	2AZ21	B;ANK
1	0AZ4	0DW46	0EV1	0EV3	1AZ5	2AZ2	2AZ21	B;ANK
4	2AZ7	2DW40	2DW44	2EV10	2EV3	2EV30	2EV34	2EV36
4	2AZ7	2DW40	2DW44	2EV10	2EV3	2EV30	2EV34	2EV36
4	2AZ7	2DW40	2DW44	2EV10	2EV3	2EV30	2EV34	2EV36
7	2EV44	BLANK	BLANK	2JQ6	2JQ8	3AZ4	3EV30	3JQ1
7	2EV44	BLANK	BLANK	2JQ6	2JQ8	3AZ4	3EV30	3JQ1
7	2EV44	BLANK	BLANK	2JQ6	2JQ8	3AZ4	3EV30	3JQ1
10	4DW11	4DW14	4EV14	BLANK	4JQ12	6JQ16	8EV24	9AZ10
10	4DW11	4DW14	4EV14	BLANK	4JQ12	6JQ16	8EV24	9AZ10
10	4DW11	4DW14	4EV14	BLANK	4JQ12	6JQ16	8EV24	9AZ10
13	9AZ7	9AZ81	9DW32	9DW37	9EV2	9EV41	9JQ5	JQ1
13	9AZ7	9AZ81	9DW32	9DW37	9EV2	9EV41	9JQ5	JQ1
13	9AZ7	9AZ81	9DW32	9DW37	9EV2	9EV41	9JQ5	JQ1
16	9GT46	352	356	0HS3	9AZ5

Gel Map:

Failed: 0EV3, 2AZ7, 2DW44, 2EV3, 2EV30, 2JQ6, 2JQ8, 3AZZ4, 4DW14, 8EV24, 9JQ5, JQ1, 2AZ2, 4EV14, 4JQ12, 6JQ16, 9AZ10

The next step is to use “cleaned DNA” Matias made for tricky samples