2.7 | Southern blot hybridization analysis
In Southern blot hybridization analysis, the 287 bp host DNA probe was
amplified by PCR of AcP13 viral DNA using 287probe-F (287probe-F 5’-CGA
TTC GTT GCG GAT G-3’) and 287-R primers (5’-CCC ATT TTG GTG CAC TTT
TA-3’) and a band corresponding to the 287 bp size was cut out for gel
extraction and labeling. The 287 bp host DNA probe was biotinylated
using the NEBlot Phototope Kit (NEB; Ipswich, MA). The transferred nylon
membrane was hybridized with the biotinylated probe for 18 hrs at 65 °C
in a hybridization solution (1X saline-sodium citrate [SSC], 0.5%
sodium dodecyl sulfate [SDS], 1X Denhardt’s). The membrane was
washed with 2× SSC and 0.1% SDS for 30 min. at 65 °C. The probe was
detected as per manufacturer’s instructions using the BCIP/NBT Color
Development Substrate kit (Promega, Madison, WI).