Table 1 PCR coanditions and primers for the detection of KHV
| Â | Gilad | Gray | TK |
|---|---|---|---|
Primer sequences | 5'-GACGACGCCGGA GACCTTGTG-3' 5'-CACAAGTTCAGTCT GTTCCTCAAC-3' | 5'-GACACCACATCTGCAA GGAG-3' 5'-GACACATGTTACAAT GGTGGC-3' | 5'-GGGTTACCTGTACGAG-3' 5'-CACCCAGTAGAT TATGC-3' |
Fragment size | 484 bp | 290 bp | 409 bp |
Enzyme | 1 unit Taq DNA polymerase platinum (Invitrogen) | 1 unit Taq DNA polymerase (Promega) | 1 unit Taq DNA polymerase platinum (Invitrogen) |
Reaction buffer | *Tris-HCl 20 nM pH = 8.4 KCl 50 mM MgCl2 2 mM (NH4)2SO4 15 mM *Taq platinum buffer | Tris-HCl 60 nM pH = 8.3 MgCl2 2 mM (NH4)2SO415 mM | *Tris-HCl 20 nM pH = 8.4 KCl 50 mM MgCl2 1.5 mM *Taq platinum buffer |
Primers / dNTP's | 30 pmole /reaction 400 μM | 0.25 μg/reaction 10 μM | 0.4 μg/reaction 200 μM |
DNA concentration | 157 ng/reaction) | 157 ng/reaction | 157 ng/reaction |
PCR reaction volume | 50 μl | 50 μl | 50 μl |
Program | 95°c for 5 min 94°c for 1 min 68°c for 1 min 72°c for 30 sec 39 cycles 72 C for 7 min | 94°c for 1 min 55°c for 2 min 72°c for 3 min 30 cycles 72°c for 7 min | 95°c 5 min 95°c 30 sec 52°c 30 sec 72°c 1 min 35 cycles 72°c for 10 min |
PCR apparatus | Biometra -T Gradient | Biometra -T Gradient | Biometra -T Gradient |