ITS2 PCR 3/1/19
E9 E10 E11 E12 E13 E14
Procedure
Samples E9 E10 E11 E12 E13 E14 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 |
|---|---|
| Water | 57 micoleters |
| 2x mytaq mix | 75 micoleters |
| ITS2 Primer Reverse (concentration: 10 micromolar) | 6 micoleters |
| ITS2 Primer Forward (concentration: 10 micromolar) | 6 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 | - |
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 1.5% 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 E9, E10, E11, E12, E13, E14 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 | E9 | E10 | E11 | Control | E12 | E13 | E14 | Control |
The voltage was set to 100V and run for 30 minutes
This is the resulting Gel
Notes
E13 and E14 were successful
E10, E11, E12 did not show the band where ITS2 should be
*E11 and E12 have an extremely faint band
E9 and the two controls did not appear at all