E16 E17 E19 E24 E30 E32 / E34 E35 E48 E52 E53 P15

Procedure

• Primer info

Samples E16 E17 E19 E24 E30 E32 E34 E35 E48 E52 E53 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 negative controls 12.5 micoleters water and 12.5 micoleters mytaq mix were added for the positive control E1 was used

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

The samples were then placed in the thermocycler set to

The number of cycles was increased to 40

Step	Temperature	Duration	cycles
1	92°C	3:00	1
2	92°C	0:30	40
3	52°C	0:40	40
4	72°C	0:30	40
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

2g agarose and 100ml 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 E16 E17 E19 E24 E30 E32 E34 E35 E48 E52 E53 P15 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	E16	E17	E19	Negative Control	E24	E30	E32	Positive Control (E1)
Ladder	E34	E35	E48	Negative Control	E52	E53	P15	Positive Control (E1)

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

This is the resulting Gel

Notes

This PCR used a positive control. having both positive and negative controls makes it easier to understand what happened if something went wrong

In this PCR the cycles were increased to 40 in hopes of amplifiing the samples that have not been amplifying.