E41 E42 E43 E44 E45 E46 E47 E48 E49 E50 E51 E52 E53 P15

Procedure

PsbA-NC is a plastid primer - Primer info

Samples E41 E42 E43 E44 E45 E46 E47 E48 E49 E50 E51 E52 E53 P15 were used in this PCR (polimerase chain reaction) reaction

Two master mixes were made A Master Mix of water, 2x mytaq mix, 1,788F Primer (concentration: 10 micromolar), 2,218R Primer (concentration: 10 micromolar)

This Master Mix x4.5

Component	Volume for 14 reactions(+5% error)
Water	132.3 micoleters
2x mytaq mix	183.75 micoleters
1,788F Primer (concentration: 10 micromolar)	14.7 micoleters
2,218R Primer (concentration: 10 micromolar)	14.7 micoleters
BAS (Borine Serum Albumin)	1 microliter

24 micoleters of this master mix was added to 7 tubes For the 2 negative controls of 25 micoleters water no positive control was available

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	63	1:30	1
3	72°C	2:00	1
4	94°C	0:45	36
5	63	1:00	36
6	72°C	1:30	36
7	72°C	2:00	1
8	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 E47 E48 E49 E50 E51 E52 E53 P15 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	P15
Ladder	E47	E48	E49	Negative Control	E50	E51	E52	E53

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

The Gels have not been run yet for this PCR

Notes

This PCR was done with the other PCRs from E15-E54 and P1-P16

instead of a positive control E53 and P15 were added to the end of these tubes