E37 E40 E41 E42 E43 E44 / E45 E46 E47 E48 E49 E50

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 E37 E40 E41 E42 E43 E44 E45 E46 E47 E48 E49 E50 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

1.5 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	-

---

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 E37 E40 E41 E42 E43 E44 E45 E46 E47 E48 E49 E50 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	E37	E40	E41	Control	E42	E43	E44	Control
Ladder	E45	E46	E47	Control	E48	E49	E50	Control

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

This is the resulting Gel

Notes