Appendix A (Table A1). A table showing Thermocycler profiles, PCR reagents, and multiplex combinations for Camp Creek coastal cutthroat trout DNA amplification.
|
Multiplex |
Loci |
Thermocycler Steps |
PCR Reagents† |
||||||
|
1 |
2 |
3 |
Primer (mM) |
MgCl2 (mM) |
dNTPs (mM) |
Promega® PCR buffer (µL) |
Tris/KCl buffer (µL) |
||
|
A |
Ots-209 |
1 cycle @ (94°C for 3 min) |
32 cycles @ (94°C for 30s + 63°C for 20s + 72°C for 30s) |
1 cycle @ 72°C for 10 min |
0.075 |
2.0 |
0.125 |
0.5 |
0.0 |
|
Ots-212 |
0.500 |
||||||||
|
B |
One-102 |
see Olsen et al. 2000‡ |
0.025 |
2.0 |
0.200 |
0.0 |
0.5 |
||
|
One-103 |
0.025 |
||||||||
|
One-108 |
0.025 |
||||||||
|
NA |
Omy-1046 |
1 cycle @ (94°C for 3 min) |
29 cycles @ (94°C for 30s + 55°C for 20s + 72°C for 30s) |
1 cycle @ 72°C for 10 min |
0.200 |
1.5 |
0.200 |
0.5 |
0.0 |
|
NA |
Ots-9 |
1 cycle @ (94°C for 3 min) |
32 cycles @ (94°C for 30s + 63°C for 20s + 72°C for 30s) |
1 cycle @ 72°C for 10 min |
0.200 |
2.0 |
0.125 |
0.5 |
0.0 |
|
NA |
Ots-10 |
1 cycle @ (94°C for 3 min) |
32 cycles @ (94°C for 30s + 63°C for 20s + 72°C for 30s) |
1 cycle @ 72°C for 10 min |
0.20 |
2.0 |
0.125 |
0.5 |
0.0 |
† All PCR reactions were conducted in 5-µL volumes using 1-µL genomic DNA template, 0.25-µL BSA PCR enhancer, and 0.025 U Taq DNA polymerase.
‡ Olsen, J. B., S. L. Wilson, E. J. Kretschmer, K. C. Jones, and J. E. Seeb. 2000. Characterization of 14 tetranucleotide microsatellite loci derived from sockeye salmon. Molecular Ecology 9:2185–2187.