Testing Diluting primers

Procedure

PsbA-NC is a plastid primer - Primer info

Samples E1 E2 E3 E4 E5 E6 E7 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: 1 and 5 micromolar), 2,218R Primer (concentration: 1 and 5 micromolar)

Component	Volume for 7 reactions(+5% error)
Water	66.15 micoleters
mytaq mix	91.9 micoleters
1,788F Primer (concentration: 1 micromolar)	7.35 micoleters
2,218R Primer (concentration: 1 micromolar)	7.35 micoleters
BAS (Borine Serum Albumin)	1 microliter

Component	Volume for 7 reactions(+5% error)
Water	66.15 micoleters
mytaq mix	91.9 micoleters
1,788F Primer (concentration: 5 micromolar)	7.35 micoleters
2,218R Primer (concentration: 5 micromolar)	7.35 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	64	1:30	1
3	72°C	2:00	1
4	94°C	0:45	36
5	64	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 E1 E2 E3 E4 E5 E6 E7 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	E1	E2	E3	Negative Control	E4	E5	E6	E7
Ladder	E1	E2	E3	Negative Control	E4	E5	E6	E7

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

This is the resulting Gel

Notes

this was to try to get rid of the secound band on all the previous PCRs