E9 E28 E30 E32 E35 E36 / E37 E44 E45 E53 P2 P14

Procedure

• Primer info

Samples E9 E28 E30 E32 E35 E36 E37 E44 E45 E53 P2 P14 were used in this PCR (polimerase chain reaction) reaction

First A Master Mix of water, 2x mytaq mix, B7Sym15 Primer Reverse (concentration: 10 micromolar), B7Sym15 Primer Forward (concentration: 10 micromolar)

Component	Volume for 14 reactions(+5% error)
Water	132.3 micoleters
2x mytaq mix	183.75 micoleters
B7Sym15 Primer Reverse (concentration: 10 micromolar)	14.7 micoleters
B7Sym15 Primer Forward (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 for the positive control E2 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 45!

Step	Temperature	Duration	cycles
1	94°C	2:00	1
2	94°C	0:30	45
3	59°C	0:60	45
4	72°C	0:15	45
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 E9 E28 E30 E32 E35 E36 E37 E44 E45 E53 P2 P14 and the two 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	E9	E28	E30	Negative Control	E32	E35	E36	Positive Control (E2)
Ladder	E37	E44	E45	Negative Control	E53	P2	P14	Positive Control (E2)

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

This is the resulting Gel

Notes

Some of E30 missed the well when being put into gel, so only a very small amount actually was in the gel which could be why E30 did not show up

In this PCR the cycles were increased to 45 in hopes of amplifiing the samples that have not been amplifying.