Fire reader system

The agarose gel was prepared in the concentration of 1%, by diluting 0.4 g of agarose in 40 mL of TAE 1x buffer (40 mM Tris base, 20 mM acetic acid, 1 mM EDTA at pH 8.0), with the addition of 0.6 μL of green safe. An amount of 18 μL of each sample and 2 μL of loading buffer was added inside the well. The electrophoresis ran at 120 V for 40 min and the gel analyses were made through Uvitec Fire-Reader system (UVItec Limited, Cambridge, UK).

Qualitative evaluation of E7 mRNA transcripts was conducted by RT-PCR. For the PCR experiment, a mixture of 0.7 μL of magnesium chloride (MgCl₂), 0.25 μL of deoxynucleotides (dNTPs), 0.40 μL of forward primer (5′−AAT CTA GAA TGC CTG ATA CAC CTA C −3′) and reverse primer (5′ −ATG GAT CCT TAT GGT TTC TGA GAA CAG A −3′), 1.25 μL of buffer, 1 μL of the cDNA sample synthesized as mentioned above, 0.25 μL of GRS Taq DNA polymerase and 8.25 μL of RNase free water. Samples were then placed into the T100™ Thermal Cycler, with the subsequent settings: 95 °C for 5 min, 26 cycles of 30 s at 95 °C, 30 s at 60 °C and 1 min at 72 °C, finally 10 min at 72 °C and to finish the amplification the samples were put into 4 °C. Final samples were analyzed by electrophoresis and visualized in the Uvitec Fire-Reader system.

The agarose gel, 1% (w/v), was prepared in 50 mL 1× TAE buffer (40 mM Tris base, 20 mM acetic acid, 1 mM EDTA at pH 8.0) and stained with GreenSafe (0.6 μL). Electrophoresis ran for 40 min at 120 V and the gel was visualized with the Uvitec Fire-Reader system (Uvitec Limited, Cambridge, UK).