PsbA-NC PCR #5 8/12/19 - Temperature Gradient
E6 E7
Procedure
Samples E6 E7 were used in this PCR (polimerase chain reaction) reaction
First A Master Mix of water, 2x mytaq mix, 1788F Primer (concentration: 10 micromolar), 2218R Primer (concentration: 10 micromolar)
| Component | Volume for 14 reactions(+5% error) |
|---|---|
| Water | 132.3 micoleters |
| mytaq mix | 183.7 micoleters |
| 1,788F Primer (concentration: 10 micromolar) | 14.7 micoleters |
| 2,218R 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 there was no positive control was available
1 microleter of the DNA samples was added to the coresponding tube
This PCR was set to 30 cycles Previous PCRs were showing up smeared and unclear. A temperature gradient was set up to try to determine a temperature where PsbA-NC works best
| Step | Temperature | Duration | cycles |
|---|---|---|---|
| 1 | 94°C | 2:00 | 1 |
| 2 | Gradient | 1:30 | 1 |
| 3 | 72°C | 2:00 | 1 |
| 4 | 94°C | 0:45 | 36 |
| 5 | Gradient | 1:00 | 36 |
| 6 | 72°C | 1:30 | 36 |
| 7 | 72°C | 2:00 | 1 |
| 8 | 4°C | hold | - |
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
| 60.3°C | 61.9°C | 63.8°C | 65.7°C | 67.6°C | 69.2°C | 70.7°C | control |
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 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 E7 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 | E6 | E6 | E6 | E6 | E6 | E6 | E6 | E6 |
| Ladder | E7 | E7 | E7 | E7 | E7 | E7 | E7 | E7 |
The voltage was set to 100V and run for 60 minutes
This is the resulting Gel
