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For PCR amplification of metazoan invertebrate mitochondrial cytochrome c oxidase subunit I (COI) gene nucleotides 1490–2198.
Forward primer: 5’•GGTCAACAAATCATAAAGATATTGG•3’
Reverse primer: 5’•TAAACTTCAGGGTGACCAAAAAATCA•3’
Materials
- Promega GoTaq® Flexi DNA polymerase
- NEB 100 bp ladder
- 1% agarose gel in TBE
Extension temperature is dependent on polymerase
| Step | Temperature (°C) | Time |
| initial denaturation | 94 | 30 sec. |
| 20 cycles | 94 | 30 sec. |
| 56◺46 | 30 sec. | |
| 72 | 1 min. | |
| 20 cycles | 94 | 30 sec. |
| 46 | 30 sec. | |
| 72 | 1 min. | |
| extension | 72 | 10 min. |
| hold | 10 | ∞ |
