To identify phaZ and bdhA in strains with PHB degradation activity, universal primers for Bacillus species were designed for each gene. MEGA-X was used to align phaZ sequences from 29 Bacillus strains and bdhA sequences from 35 Bacillus strains. Among the aligned sequences, primers were designed by identifying the conserved regions common to most strains. Primer sequences used to identify phaZ using PCR were forward (5’-AATAAGTGTTGGAACTGGTTTGA-3’) and reverse (5’-GTGTCGGTATTGTACATTCCTTTATC-3’). The primer sequences used to identify bdhA by PCR were forward (5’-ATTGAAGAATTTCCTACAGAA-3’) and reverse (5’-TGGAGTATCCACATAACCAGGGCA-3’). The PCR conditions for phaZ and bdhA used in this study are listed in Table 1 . The polymerase used for PCR was 2X Pfu PCR Smart Mix 1 (Solgent, Korea). PCR products were separated using electrophoresis at 100 mV for 30 min on 1.5% agarose gel containing 4 μl EcoDye DNA Staining Solution (Solgent) in TAE buffer (Bioneer, Korea).
Tae buffer
Manufactured by Bioneer
TAE buffer is a commonly used buffer solution in molecular biology and biotechnology laboratories. It is primarily used as an electrophoresis buffer for the separation and analysis of nucleic acids, such as DNA and RNA, in agarose gel electrophoresis.
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2 protocols using tae buffer
1
Bacillus phaZ and bdhA Gene Detection
2
SSR-Based DNA Profiling Protocol
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Eight pairs of SSR primers were tested for amplification and among them only five primers were found good for DNA profiling (Table 2) based on readable and reproducibly of the bands. SSR amplification was performed in 20 µl reaction volume containing 50ng genomic DNA, 10 µl of 2X Green GoTaq® Reaction Buffer (pH 8.5), 400μM dATP, 400μM dGTP, 400μM dCTP, 400μM dTTP and 3 mM MgCl2 (Promega) and 10 picomole of each forward and reverse primer. These components were gently mixed and centrifuged prior to adding 2 drops of mineral oil. The amplification was performed in a Thermo cycler (MULTIGENE OPTIMAX, Labnet International, Inc.). The cycling conditions were 1 cycle of 95°C for 5 min followed by 30 cycles of 95°C for 60 sec, 55°C for 1 min 30 sec, 72°C for 2 min, and finally 1 cycle of 72°C for 10 min.
After finishing amplification 12 µl aliquots of amplification products was loaded in a 1% (w/v) agarose gel (Bioneer) for electrophoresis in 1X TAE buffer (Bioneer). After completion of electrophoresis, gels were stained with Ethidium bromide (0.5 µg/ml for 30 min) and visualized under exposure of UV light within gel doc system (UVDI, Major Science). There was standard 100 bp ladder (Promega) for comparing size of the band.
After finishing amplification 12 µl aliquots of amplification products was loaded in a 1% (w/v) agarose gel (Bioneer) for electrophoresis in 1X TAE buffer (Bioneer). After completion of electrophoresis, gels were stained with Ethidium bromide (0.5 µg/ml for 30 min) and visualized under exposure of UV light within gel doc system (UVDI, Major Science). There was standard 100 bp ladder (Promega) for comparing size of the band.
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