Mastercycler pcr instrument

A 77-base-pair-long 5′-amino-DNA to be conjugated to secondary antibody was 5′-end aminated via PCR using synthetic oligonucleotide as a template and forward primers containing an amino group at its 5′ end. The sequence of the synthetic oligonucleotide serving as a template was: 5′-TCCGGTCGCTATCGTTTGAAAGTCGAGGGCGACCACGAGGAGGAGGTCTGCGAGGTAGCGTTAATCGAGAGCAGTGA-3′. The sequences of forward and reverse primers were 5′-TCCGGTCGCTATCGTTTGAA-3′ and 5′-TCACTGCTCTCGATTAACGCT-3′, respectively. The PCR mixture contained 1× Taq buffer with (NH₄)₂SO₄, 3 mM MgCl₂, 0.3 mM each primer, 0.2 mM each dNTP, 2.5 U Taq DNA polymerase, and nuclease-free H₂O in a total volume of 50 µL. The amplification reaction conditions included denaturation at 95 °C for 4 min, followed by 40 cycles of 95 °C for 20 s, 56 °C for 30 s, and 72 °C for 30 s. The final extension was performed at 72 °C for 5 min. PCR amplification was performed using Mastercycler PCR instrument (Eppendorf; Hamburg, Germany). PCR-amplified amino-DNA was pooled and purified using a PCR clean-up kit. Following purification, amino-DNA was first freeze-dried and then covalently conjugated to secondary unconjugated goat anti-rabbit antibodies using a commercially available oligonucleotide conjugation kit by following the manufacturer’s instructions. The molar ratio of DNA to antibodies was 3:1.

To verify whether the typing PCR was feasible. The reaction components for the PCR assays were as follows:12.5 μl PCR Mix, 10 μl dd H₂O, 0.5 μl HSV-1 template or HSV-2 template, 0.5 μl primer 1-F, 0.5 μl primer 1-R, 0.5 μl primer 2-F, 0.5 μl primer 2-R. It was amplified in Eppendorf Master cycler PCR instrument (Eppendorf, Hamburg, Germany) for predegeneration at 95°C for 5 min, and then 35 cycles at 95°C 30 s, 58°C for 30 s, and 72°C 30 s, followed by a final extension at 72°C for 10 min. Finally, the PCR products were analyzed by 1% agarose gel.