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Table 1 PCR coanditions and primers for the detection of KHV

From: Cloning of the koi herpesvirus (KHV) gene encoding thymidine kinase and its use for a highly sensitive PCR based diagnosis

  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