|
PCR coanditions and primers for the detection of KHV |
|||
| 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 |
Bercovier et al. BMC Microbiology 2005 5:13 doi:10.1186/1471-2180-5-13 |
|||