E53 E54 P13 P14 P15 P16 E4 E11 E25 E27 E30 E43

Procedure

• Primer info

Samples E53 E54 P13 P14 P15 P16 E4 E11 E25 E27 E30 E43 were used in this PCR (polimerase chain reaction) reaction

First A Master Mix of water, 2x mytaq mix, CA4.86R Primer (concentration: 10 micromolar), CA4.86L Primer (concentration: 10 micromolar)

Component	Volume for 14 reactions(+5% error)
Water	132.3 micoleters
2x mytaq mix	183.75 micoleters
CA4.86R Primer (concentration: 10 micromolar)	14.7 micoleters
CA4.86L Primer (concentration: 10 micromolar)	14.7 micoleters

24 micoleters of this master mix was added to 28 tubes For the 4 negative controls 12.5 micoleters water and 12.5 micoleters mytaq mix were added E2 was used as 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 the annealing temperature was changed from 50°C to 52°C to see if this would get rid of the extra band showing up in the Gels

Step	Temperature	Duration	cycles
1	92°C	3:00	1
2	92°C	0:30	35
3	52°C	1:00	35
4	72°C	0:15	35
5	72°C	2:00	1
6	4°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 E53 E54 P13 P14 P15 P16 E4 E11 E25 E27 E30 E43 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	E53	E54	P13	Negative Control	P14	P15	P16	Positive Control (E2)
Ladder	E4	E11	E25	Negative Control	E27	E30	E43	Positive Control (E2)

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

This is the resulting Gel

Notes

The first set of PCRs was finishing the first go through of samples. The secound set is redos of PCRs that did not work the first time