E8 E9 E10 E16 E17 E19 E20 / E21 E24 E28 E30 E32 P14 P15

Procedure

• Primer info

PCR was performed on samples that have not been used before, these samples are from aliquoites of DNA extractions becuase the original coral samples are not available

Samples E8 E9 E10 E16 E17 E19 E20 E21 E24 E28 E30 E32 P14 P15 were used in this PCR (polimerase chain reaction) reaction

First A Master Mix of water, 2x mytaq mix, ITS2 Primer Reverse (concentration: 10 micromolar), ITS2 Primer Forward (concentration: 10 micromolar)

Component	Volume for 14 reactions (+5% error)
	Water	132.3 micoleters
	2x mytaq mix	183.75 micoleters
	ITS2 Primer Reverse (concentration: 10 micromolar)	14.7 micoleters
	ITS2 Primer Forward (concentration: 10 microolar)	14.7 micoleters

24 micoleters of this master mix was added to 14 tubes For the 2 controls 12.5 micoleters water and 12.5 micoleters mytaq mix were added

1 microleters of the DNA samples was added to the coresponding tube and 1 microleter of water was added to two tubes for the controls

The samples were then placed in the thermocycler set to

Step	Temperature	Duration	cycles
1	92°C	3:00	1
2	92°C	0:30	35
3	52°C	0:40	35
4	72°C	0:30	35
5	72°C	5:00	1
6	10°C	hold	-

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these amplified DNA samples were then tested by making a gel

to create the Gel the following steps were taken

1.5g agarose and 75ml 1X TAE buffer were mixed to make a 2% agarose 75ml gel mix the mix was microwaved and swirled until there were no flakes then 1 microliter Gelgreen was added

the mix was left to cool for 5 minutes then poured into the gel mold the gel was left to harden for 30 minutes

once hardened enough Used TAE buffer was poured into the gel box to cover the mold

5 microliter aliquots of DNA samples E8 E9 E10 E16 E17 E19 E20 E21 E24 E28 E30 E32 P14 P15 and the two controls were made 1 microliter of 5x loading dye was added to each

First the HyperLadder 100bp was added to the first well of the mold then the DNA samples were each added to the wells in the following order

Ladder	E8	E9	E10	Control	E16	E17	E19	E20
Ladder	E21	E24	E28	Control	E30	E32	P14	P15

The voltage was set to 100V and run for 60 minutes

This is the resulting Gel

Notes

New Primers, Extractions, Water, and biomix were used in this PCR