ITS amplicon protocol
PCR specifications
Target Region and Size: ITS 1 region (~321 bp)
Template Concentration: 50 ng
Enzyme and Master mix: TaKaRa LA Taq (Cat. No. RR002A)
Primers
| ID | Primer sequence | Length |
|---|---|---|
| ITS1F (1723-1744) | TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCTTGGTCATTTAGAGGAAGTAA | 55 |
| ITS2R (2024-2043) | GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCTGCGTTCTTCATCGATGC | 54 |
PCR Mixture
| Reagents | Concentration | 1 reaction (25 μL) |
|---|---|---|
| DNA Template | 50 ng | 1.7 μL |
| 10X LA PCR Buffer | 1X | 2.5 μL |
| MgCl2 | 1.8 mM | 1.8 μL |
| dNTPs | 0.1 mM | 1 μL |
| Forward Primer | 0.28 pmol | 0.7 μL |
| Reverse Primer | 0.28 pmol | 0.7 μL |
| BSA | 0.1 μg | 0.5 μL |
| Nuclease Free Water | - | 15.975 μL |
| Takara LA Taq | 0.625 U | 0.125 μL |
| Total | - | 25 μL |
PCR Conditions
| Step | Temperature | Time | Cycle |
|---|---|---|---|
| 1 | 95 °C | 5 minutes | 1 |
| 2 | 95 °C → 56 °C → 72 °C | 30 sec → 30 sec → 30 sec | 32 |
| 3 | 72 °C | 7 minutes | 1 |
| 4 | 20 °C | ∞ | - |