PCR 1 amplifying ITS2 marker for Box 1 - O.Fav deep

Protocol

NEW PCR PROTOCOL – multiple failed amplifications (see ITS2_PRC1_7-11) led to re evaluation of Master Mix concentrations 1.0 uL DNA –> 3.0 uL DNA 12.5 uL MyTaq –> 25 uL Mytaq 1.0 uL Primer –> 2.0 uL Primer

Samples:

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

Species	Treatment	ID
OFAV	P	9GT4
OFAV	P	9GT46
OFAV	P	9IR44
OFAV	P	9JQ4
OFAV	P	9JQ42
OFAV	P	9JQ431
OFAV	P	9JQ53
OFAV	P	AZ
OFAV	P	2HS12
OFAV	P	9AZ5
OFAV	P	9AZ9
OFAV	P	9EV35
OFAV	P	9EV4
OFAV	P	9EV40
OFAV	P	9EV5
OFAV	P	9GT35

Master Mix:

Component	Volume for 1 rxn	Volume for 16 rxns (+10% error)
2x MyTaq Mix	25.0 uL	440.0 uL
Molecular Water	18.0 uL	316.8 uL
ITS2 Primer Forward (10 uM)	2.0 uL	35.0 uL
ITS2 Primer Reverse (10 uM)	2.0 uL	35.0 uL
DNA	3.0 uL	NA

Strip Tube Set Up:

Strip (replicate) Number	A	B	C	D	E	F	G	H
1	9GT4	9GT46	9IR44	9JQ4	9JQ42	9JQ431	9JQ53	AZ
2	9GT4	9GT46	9IR44	9JQ4	9JQ42	9JQ431	9JQ53	AZ
3	9GT4	9GT46	9IR44	9JQ4	9JQ42	9JQ431	9JQ53	AZ
4	2HS12	9AZ5	9AZ9	9EV35	9EV4	9EV40	9EV5	9GT35
5	2HS12	9AZ5	9AZ9	9EV35	9EV4	9EV40	9EV5	9GT35
6	2HS12	9AZ5	9AZ9	9EV35	9EV4	9EV40	9EV5	9GT35

Gel Map:

Failed: 9AZ5 (x3) All others passed