51°C annealing temp: E41 E42 E43 E44 E45 E46 P1 P2 P3 P4 P5 P6

Procedure

• Primer info

Samples E41 E42 E43 E44 E45 E46 P1 P2 P3 P4 P5 P6 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 CA4 setting the annealing temperature was changed from 50°C to 51°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	51°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 E41 E42 E43 E44 E45 E46 P1 P2 P3 P4 P5 P6 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	E41	E42	E43	Negative Control	E44	E45	E46	Positive Control (E2)
Ladder	P1	P2	P3	Negative Control	P4	P5	P6	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 anealing temperature of 51°C they worked well and the secound band that was showing up in previous PCRs seemed to have gone away

This Gel had slightly more clear results than the gel with a higher annealing temperature so 51°C is what I will use going forward.