E13 E14 E15 E16 E17 E18 / P1 P2 P3 P4 P5 P6

Procedure

• Primer info

A new set of B7Sym15 Primers were created to test if this was the reason no DNA showed up in the last test.

Samples E13 E14 E15 E16 E17 E18 P1 P2 P3 P4 P5 P6 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 12 reactions(+5% error)
Water	113.4 micoleters
2x mytaq mix	157.5 micoleters
B7Sym15 Primer Reverse (concentration: 10 micromolar)	12.6 micoleters
B7Sym15 Primer Forward (concentration: 10 micromolar)	12.6 micoleters

24 micoleters of this master mix was added to 12 tubes For the 4 controls 12.5 micoleters water and 12.5 micoleters mytaq mix were added

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	94°C	2:00	1
2	94°C	0:15	32
3	59°C	0:15	32
4	72°C	0:15	32
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

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 E13 E14 E15 E16 E17 E18 P1 P2 P3 P4 P5 P6 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	E13	E14	E15	Control	E16	E17	E18	Control
Ladder	P1	P2	P3	Control	P4	P5	P6	Control

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

These are the resulting Gels

Notes