E1 E2 E3 E4 E5 E6 / E7 E8 E9 E10 E11 E12

Procedure

• Primer info

Samples E1 E2 E3 E4 E5 E6 E7 E8 E9 E10 E11 E12 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

The denaturing, Annealing, and Extension times were changed from 15 seconds to 30/60/15 and the cycles were increased to 35

Step	Temperature	Duration	cycles
1	94°C	2:00	1
2	94°C	0:30	35
3	59°C	0:60	35
4	72°C	0:15	35
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 E1 E2 E3 E4 E5 E6 E7 E8 E9 E10 E11 E12 and the four 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	E1	E2	E3	Control	E4	E5	E6	Control
Ladder	E7	E8	E9	Control	E10	E11	E12	Control

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

This is the resulting Gel

Notes

a band showed up for all samples, some were just extremely faint while not perfect yet this solution fixed the issue with the B7sym15 PCRs possibly adding more cycles could bring out the very faint samples

The length of the bands do not match up with the expected B7sym15 length due to them having an attached tail to aid in sequencing them.