This experiment is to verify fullITS primers can be sequenced. Primer Source Paper
Primer bases:
ITS-p5 LCO1490F (forward): CCTTATCAYTTAGAGGAAGGAGGGTCAACAAATCATAAAGATATTGG
ITSCOI-p4 CFMRa (reverse): CCGCTTAKTGATATGCTTAAA94 °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. GGWACTAATCAATTTCCAAATCC
95 °C for 60 s, 45 °C for 90 s, 72 °C for 90 s; 28 cycles of: 94 °C for 60 s, 50 °C for 90 s, 72 °C for 60 s
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 |
Add 11 ul to each well of a hard shell, full skirt plate. Add 2 uL of 0.5 uM primers and 2uL of template to each well.
Primers:
Run fullITS CO1Lark plates on thermocycler program fullITSCO1Lark:
Temp C
Cycles
Time
95*
1X
3:00*
98
35X
0:30
62
35X
0:30
72
35X
0:30
72
1X
Step | Temp C | Cycles | Time |
|---|---|---|---|
Denature | 95 | 1X | 10:00 |
Denature | 94 | 35X | 01:3000 |
Annealing** (Row C) | 5550 | 35X | 01:4030 |
Extension/Elongation | 72 | 35X | 1:00 |
Final Extension | 72 | 1X | 10:00 |
Hold | 4 | 1X | 0:00 |
Run 16S plates on Thermocycler Program GSAF35:
4 | 1X | 0:00 |
Pool duplicates together.
...