E53 E54 P13 P14 P15 P16

Procedure

24F is also known as cp23S-rDNA and is a chloroplast primer - Primer info

Samples E53 E54 P13 P14 P15 P16 were used in this PCR (polimerase chain reaction) reaction

First A Master Mix of water, 2x mytaq mix, 24F Primer (concentration: 10 micromolar), 454R Primer (concentration: 10 micromolar)

Component	Volume for 14 reactions (+5% error)
Water	132.3 micoleters
2x mytaq mix	183.75 micoleters
24F Primer (concentration: 10 micromolar)	14.7 micoleters
454R Primer (concentration: 10 micromolar)	14.7 micoleters
BAS (Borine Serum Albumin)	1 microliter

24 micoleters of this master mix was added to 14 tubes For the 2 negative controls 12.5 micoleters water and 12.5 micoleters mytaq mix were added E3 was used as a 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 cycless were changed to 30 and the temperature changed to 55.5°C to get rid of the secound band

Step	Temperature	Duration	cycles
1	92°C	3:00	1
2	92°C	0:30	30
3	55.5°C	1:00	30
4	72°C	0:15	30
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 large 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 E53 E54 P13 P14 P15 P16 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

E53

E54

P13

Negative Control

P14

P15

P16

Positive Control (E3)

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

This is the resulting Gel

Notes

finishing 24F/454R primer

This primer was a algal primer which is an explination for why many samples worked that had not worked before.

this primer is also called cp23S-rDNA