Test ITS2 PCR on: P1 P3 P4 E34 E35 E36

Procedure

• Primer info

PCR was performed on samples that have not been used before, these samples are from aliquoites of DNA extractions becuase the original coral samples are not available

Samples P1 P3 P4 E34 E35 E36 were used in this PCR (polimerase chain reaction) reaction

First A Master Mix of water, 2x mytaq mix, ITS2 Primer Reverse (concentration: 10 micromolar), ITS2 Primer Forward (concentration: 10 micromolar)

Component	Volume for 6 reactions(+5% error)
Water	59.85 micoleters
2x mytaq mix	78.75 micoleters
ITS2 Primer Reverse (concentration: 10 micromolar)	6.3 micoleters
ITS2 Primer Forward (concentration: 10 micromolar)	6.3 micoleters

24 micoleters of this master mix was added to 6 tubes For the 2 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 and 1 microleter of water was added to two tubes for the controls

The samples were then placed in the thermocycler set to

Step	Temperature	Duration	cycles
1	92°C	3:00	1
2	92°C	0:30	35
3	52°C	0:40	35
4	72°C	0:30	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

2g agarose and 100ml 1X TAE buffer were mixed to make a 2% agarose 100ml gel mix the mix was microwaved and swirled until there were no flakes then 5 microliters Gelred 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 P1 P3 P4 E34 E35 E36 and the two 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

P1

P3

P4

Control

E34

E35

E36

Control

The voltage was set to 100V and run for 30 minutes

This is the resulting Gel

Notes

The “P” samples came out very strong The “E” samples did not come out, this could cause problems since their is no original coral sample for these E’s