24F PCR #2 7/16/19
E15 E16 E17 E18 E19 E20 E21 E22 E23 E24 E25 E26
Procedure
24F is also known as cp23S-rDNA and is a chloroplast primer - Primer info
Samples E15 E16 E17 E18 E19 E20 E21 E22 E23 E24 E25 E26 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 | - |
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 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 E15 E16 E17 E18 E19 E20 E21 E22 E23 E24 E25 E26 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 | E15 | E16 | E17 | Negative Control | E18 | E19 | E20 | Positive Control (E3) |
| Ladder | E21 | E22 | E23 | Negative Control | E24 | E25 | E26 | Positive Control (E3) |
The voltage was set to 100V and run for 60 minutes
This is the resulting Gel
