52°C annealing temp: E47 E48 E49 E50 E51 E52 P7 P8 P9 P10 P11 P12

Procedure

• Primer info

Samples E47 E48 E49 E50 E51 E52 P7 P8 P9 P10 P11 P12 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 28 reactions(+5% error)
Water	2x 132.3 micoleters
2x mytaq mix	2x 183.75 micoleters
CA4.86R Primer (concentration: 10 micromolar)	2x 14.7 micoleters
CA4.86L Primer (concentration: 10 micromolar)	2x 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 E47 E48 E49 E50 E51 E52 P7 P8 P9 P10 P11 P12 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	E47	E28	E49	Negative Control	E50	E51	E52	Positive Control (E2)
Ladder	P7	P8	P9	Negative Control	P10	P11	P12	Positive Control (E2)

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

This is the resulting Gel

Notes

These two sets had an annealing temperature of 52°C they worked well and the secound band that was showing up in previous PCRs seemed to have gone away

however these were very blurry and so using an annealing temp of 51°C may be the best option going forward.