P14 P15 E51 E52 E53 E54 / E34 E35 E36 E37 E43 E44

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 P14 P15 E51 E52 E53 E54 E34 E35 E36 E37 E43 E44 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 12 reactions (+5% error)
	Water	113.4 micoleters
	2x mytaq mix	157.5 micoleters
	ITS2 Primer Reverse (concentration: 10 micromolar)	12.6 micoleters
	ITS2 Primer Forward (concentration: 10 micromolar)	12.6 micoleters

24 micoleters of this master mix was added to 12 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 5 microliter Gelred were 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 P14 P15 E51 E52 E53 E54 E34 E35 E36 E37 E43 E44 and the four 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	P14	P15	E51	Control	E52	E53	E54	Control
Ladder	E34	E35	E36	Control	E37	E43	E44	Control

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

This is the resulting Gel

Notes