E4 E7 E8 E9 E10 E12 E18 E25 E30 E31 E43 E54

Procedure

• Primer info

Samples E4 E7 E8 E9 E10 E12 E18 E25 E30 E31 E43 E54 were used in this PCR (polimerase chain reaction) reaction

First A Master Mix of water, 2x mytaq mix, 23S1M13 Primer (concentration: 10 micromolar), 23S2M13 Primer (concentration: 10 micromolar)

Component	Volume for 14 reactions(+5% error)
Water	132.3 micoleters
2x mytaq mix	183.75 micoleters
23S1M13 Primer (concentration: 10 micromolar)	14.7 micoleters
23S2M13 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 E2 was used for the positive control

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

The samples were then placed in the thermocycler set to run for 45 cycles

Step	Temperature	Duration	cycles
1	92°C	3:00	1
2	92°C	0:30	45
3	55°C	1:00	45
4	72°C	0:15	45
5	72°C	5:00	1
6	4°C	hold	-

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The 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 E4 E7 E8 E9 E10 E12 E18 E25 E30 E31 E43 E54 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	E4	E7	E8	Negative Control	E9	E10	E12	Positive Control (E2)
Ladder	E18	E25	E30	Negative Control	E31	E43	E54	Positive Control (E2)

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

This is the resulting Gel

Notes

Older primers were used

The thermomixer was set to 45 cycles