Primer premier 5

A pair of primers was designed by Primer premier 5.0 and synthesized by Invitrogen (USA). To facilitate cloning, HindIII or BamHI restriction sites (underlined) were incorporated into the forward primer (5'-CCAAGCTTCGATGGCTTCTATGGAGACAGC-3') and reverse primer (5'-CGGGATCCTTACCAATCTACCCTAAACATG-3'), respectively. Primers for amplifying DEV gM (forward: 5'-CTCGAGATGGGATCCATGAATGGGC-3' and reverse: (5'-CCGCGGTTCACTATCCGACGAGAAATGAGT-3') containing XhoI or SacII restriction sites (underlined), respectively, were also designed by Primer premier 5.0 and synthesized by Invitrogen (USA). Using DEV CHv DNA as the template, the PCR was performed with reactions (20 µL/tube) containing 10 µL PrimeSTAR (premix) DNA polymerase, 0.4 µL of each primer (10 pmol each), 0.3 µL DNA template, and 8.9 µL ultrapure water.
DEV UL49.5 PCR products and pEGFP-C1 were digested by HindIII or BamHI, respectively, and then ligated with T4 ligase to produce the pEGFP-C1/pUL49.5 plasmid (Fig. 1A). The pDsRed1-N1/gM plasmid was constructed in the same way following digestion by XhoI and SacII (Fig. 1B). Successful production of the pEGFP-C1/pUL49.5 and pDsRed1-N1/gM plasmids was subsequently confirmed by PCR, restriction enzyme digestion, and sequencing as previously described [6 (link)].

The total RNA were isolated from B. amyloliquefaciens cultures using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) and then treated with RNase-free DNase. First-strand cDNA was conversed from total RNA using an RT-PCR kit (Fermentas, Vilnius, Lithuania¹⁵ (link)). Real-time PCR was performed as described in the paper.¹⁶ (link) Band intensities were normalized to the 16S rDNA transcript band for 2^−ΔΔCT relative quantification. The B. amyloliquefaciens nucleotide sequences for these genes were obtained from the NCBI GenBank database. Primer pairs were designed from these sequences with Primer Premier 5.0 software (Applied Biosystems), the 16S rDNA primers used were F(5-CCTACGGGAGGCAGCAG-3) and R (5-ATTACCGCGGCT GCTGG-3), the comA primers were F (5-TCAAAGTGAGCAGGATCGGTTAA-3) and R (5-CTTCTGTACGGGAGCCGACAT-3) and spo0A primers were F (5-TTGCGGCG ATGAAGTGAATG-3) and R (5-CGATGGAAAGCTGCGGTGTA-3).