P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12

Procedure

• Primer info

Samples P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 were used in this PCR (polimerase chain reaction) reaction

First A Master Mix of water, 2x mytaq mix, ITS1 Primer (concentration: 10 micromolar), ITS1intrev2 Primer (concentration: 10 micromolar)

Component	Volume for 14 reactions(+5% error)
Water	132.3 micoleters
2x mytaq mix	183.75 micoleters
ITS1 Primer (concentration: 10 micromolar)	14.7 micoleters
ITS1intrev2 Primer (concentration: 10 micromolar)	14.7 micoleters

24 micoleters of this master mix was added to 14 tubes For the 2 negative controls 12.5 micoleters water and 12.5 micoleters mytaq mix were added E1 was used as a positive control

1 microleter of the DNA samples was added to the coresponding tube

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	59°C	1:00	35
4	72°C	0:15	35
5	72°C	5:00	1
6	4°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 100ml gel mix the mix was microwaved and swirled until there were no flakes then 1 microliters 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 P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 and the two controls were made 1 microliter of 5x loading dye was added to each

First 3 microliters of 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	P1	P2	P3	Negative Control	P4	P5	P6	Positive Control (E1)
Ladder	P7	P8	P9	Negative Control	P10	P11	P12	Positive Control (E1)

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

This is the resulting Gel

Notes

Older Primers were used for this PCR