E4 E30 E34 E54 P2 P3

Procedure

• Primer info

Samples E4 E30 E34 E54 P2 P3 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	66.15 micoleters
2x mytaq mix	91.9 micoleters
23S1M13 Primer (concentration: 10 micromolar)	7.35 micoleters
23S2M13 Primer (concentration: 10 micromolar)	7.35 micoleters

24 micoleters of this master mix was added to 14 tubes For the negative control 12.5 micoleters water and 12.5 micoleters mytaq mix were added E1 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 50 cycles

Step	Temperature	Duration	cycles
1	92°C	3:00	1
2	92°C	0:30	50
3	55°C	1:00	50
4	72°C	0:15	50
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

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 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 E30 E34 E54 P2 P3 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

E30

E34

Negative Control

E54

P2

P3

Positive Control (E1)

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

This is the resulting Gel

Notes

The thermomixer was set to 50 cycles