This experiment is to verify fullITS primers can be sequenced. Primer Source Paper
Primer bases:
ITS-p5 (forward): CCTTATCAYTTAGAGGAAGGAG
ITS-p4 (reverse): CCGCTTAKTGATATGCTTAAA
94 °C for 4 min, followed by 34 cycles of 30 s at 94 °C, 40 s at 55 °C (or 58 °C) and 1 min at 72 °C, with a final step of 10 min at 72 °C.
PCR MasterMix
ul/rxn | Reagent | # of rxns | ul needed |
|---|---|---|---|
3 | 5X Kapa HiFi Buffer | 384 | 1320 |
0.45 | 10M dNTPs | 384 | 198 |
0.3 | Kapa HiFi HotStart DNA Pol | 384 | 132 |
7.25 | HPLC H2O | 384 | 3190 |
11 | Total Volume | 384 | 4840 |
...
Step | Temp C | Cycles | Time |
|---|---|---|---|
Denature | 95 | 1X | 10:00 |
Denature | 9594 | 35X | 0:30 |
Annealing** (Row C) | 55 | 35X | 0:3040 |
Extension/Elongation | 72 | 35X | 01:3000 |
Final Extension | 72 | 1X | 910:00 |
Hold | 4 | 1X | 0:00 |
Run 16S plates on Thermocycler Program GSAF35:
...