B7Sym15 test #4 PCR 6/4/19 - Adding cycles to the PCR
E1 E2 E3 E4 E5 E6
Procedure
Samples E1 E2 E3 E4 E5 E6 were used in this PCR (polimerase chain reaction) reaction
First A Master Mix of water, 2x mytaq mix, B7Sym15 Primer Reverse (concentration: 10 micromolar), B7Sym15 Primer Forward (concentration: 10 micromolar)
| Component | Volume for 6 reactions(+5% error) |
|---|---|
| Water | 59.85 micoleters |
| 2x mytaq mix | 78.75 micoleters |
| B7Sym15 Primer Reverse (concentration: 10 micromolar) | 6.3 micoleters |
| B7Sym15 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
The samples were then placed in the thermocycler set to
The number of cyc;es was changed from 30 to 35
| Step | Temperature | Duration | cycles |
|---|---|---|---|
| 1 | 94°C | 2:00 | 1 |
| 2 | 94°C | 0:30 | 35 |
| 3 | 59°C | 0:30 | 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 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 E1 E2 E3 E4 E5 E6 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 | E1 | E2 | E3 | Control | E4 | E5 | E6 | Control |
The voltage was set to 100V and run for 60 minutes
This is the resulting Gel
Notes
This trial was much more succsesful and so going forward the methods of this test and test #3 will be combinded to see if that leads to greater success
the length of the bands do not match up with the expected B7sym15 length due to them having a attached tail to aid in sequencing them.