E27 E28 E29 E30 E31 E32 E33 E34 E35 E36 E37 E40

Procedure

28S is also known as ITS2_Coral and is a ITS2 primer - Primer info

Samples E27 E28 E29 E30 E31 E32 E33 E34 E35 E36 E37 E40 were used in this PCR (polimerase chain reaction) reaction

First A Master Mix of water, 2x mytaq mix, 5.8S-436 Primer (concentration: 10 micromolar), 28S-663 Primer (concentration: 10 micromolar)

Component	Volume for 14 reactions(+5% error)
Water	132.3 micoleters
2x mytaq mix	183.75 micoleters
5.8S-436 Primer (concentration: 10 micromolar)	14.7 micoleters
28S-663 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 this was the first 5.8S.38 PCR so 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	92°C	3:00	1
2	92°C	0:30	25
3	56°C	1:00	25
4	72°C	0:15	25
5	72°C	5:00	1
6	4°C	hold	-

---

these amplified DNA samples were then tested by making a gel

to create the Gel the following steps were taken

1.5g 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 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 E27 E28 E29 E30 E31 E32 E33 E34 E35 E36 E37 E40 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	E27	E28	E29	Negative Control	E30	E31	E32	Positive Control (E5)
Ladder	E33	E34	E35	Negative Control	E36	E37	E40	Positive Control (E5)

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

This is the resulting Gel

Notes

the wells in Gel #4 were messed up a little bit