Pcr xl topo vector

Full length cDNA of EV-A71 was amplified using polymerase chain reaction (PCR). Each PCR reaction contained 5X Phusion HF buffer, 10 mM dNTP, 0.1 g of each primer, 1 μg template cDNA and Phusion HSII DNA Polymerase. The following cycling conditions were employed for the PCR reactions: 98 °C for 30s, followed by 98 °C for 10s, 64 °C for 30s and 72 °C for 5 min for 30 cycles. The cycles were terminated with a 30s extension at 68 °C. The amplified cDNA was cloned into the pCR®-XL-TOPO® vector (Invitrogen, Calif., USA). Approximately 100 ng of the full length EV-A71 cDNA was ligated to 1 μL pCR®-XL-TOPO® vector according to the manufacturer’s protocol in the TOPO® XL PCR Cloning Kit (Invitrogen, Calif., USA). The ligation mixture was incubated for 5 min at room temperature, then 1 μl of the 6x TOPO® Cloning stop solution was added and the tubes were placed on ice. The recombinant EV-A71 pCR®-XL-TOPO® vector was stored at −20 °C for further downstream processes.

The BOGUAY_2967, BOGUAY_2386, and BOGUAY_0691 target genes including their flanking regions were PCR-amplified with primer combinations 2967 EXP F and R, 2386 EXP F and R, and 0691 EXPF and R (primer pairs 1–3, Table 2) from genomic amplified DNA of a single orange filament, collected during Alvin dive 4568; initial PCR amplifications with primers that excluded the flanking regions were not successful (primer pairs 4–6, Table 2). The “flanking region” PCR products were then cloned into a pCR-XL-TOPO vector (Thermo Fisher) and again PCR-amplified, now with primers 2967 F and R, 2386 F and R, and 0691 F and R (primer pairs 4–6, Table 2) located in the terminal regions of the target genes and excluding the flanking regions. These primers resulted in shorter but consistently retrieved PCR amplicons. The resulting PCR products were cloned into vector plasmid pCR2.1 (Supplementary Table 1), transformed into Escherichia coli and grown to obtain inserts without flanking regions. For subsequent gene expression, the target genes (now without flanking regions) were put into the pET22b vectors for expression in E. coli strain BL21. The primer pairs 4–6 were modified by adding different restriction sites to forward and reverse versions (primer pairs 7–9), for cloning the target genes into pET22b with correct directionality.