E53 E54 P13 P14 P15 P16 E4 E7 E34 E36 E37 P2

Procedure

• Primer info

Samples E53 E54 P13 P14 P15 P16 E4 E7 E34 E36 E37 P2 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 E53 E54 P13 P14 P15 P16 E4 E7 E34 E36 E37 P2 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	P53	P54	P13	Negative Control	P14	P15	P16	Positive Control (E1)
Ladder	E4	E7	E34	Negative Control	E36	E37	P2	Positive Control (E1)

The voltage was set to 125V and run for 45 minutes

This is the resulting Gel

Notes

The first Gel finishes the first go through of ITS1

The second Gel is doing some of the samples that did not work well the first time again