Yeasts were identified by analysis of the D1/D2 region of the
large subunit of rRNA (LSU rRNA) gene sequence similarities
(Kurtzman & Robnett 1998). Methods for DNA extraction and
amplification of the D1/D2 domain were described previously
(Limtong et al. 2007). The D1/D2 domain of the LSU rRNA gene
was PCR amplified from yeast genomic DNA. The D1/D2 region
of the LSU rRNA gene was amplified and sequenced with
primers, NL1 and NL4 (Kurtzman & Robnett 1998). The PCR
products were checked by agarose gel electrophoresis and purified using HiYieldä Gel/PCR DNA Fragments Extraction Kit
(RBC Bioscience, Chung Ho City, Taipei, Taiwan). The purified
products were submitted to Macrogen (Korea) for sequencing.
Sequencing reactions were performed in a MJ Research PTC225 Peltier Thermal Cycler, using an ABI PRISMÒ BigDyeä Terminator Cycle Sequencing Kit with AmpliTaqÒ DNA polymerase (FS enzyme) (Applied Biosystems, USA), following the
protocols supplied by the manufacturer. Single-pass sequencing was performed on each template using the primers NL1
and NL4 for the D1/D2 region of the LSU rRNA gene. The
fluorescent-labelled fragments were purified from the unincorporated terminators with an ethanol precipitation protocol. The samples were resuspended in distilled water and
subjected to electrophoresis in an ABI 3730XL sequencer (Applied Biosystems, USA). The sequences were compared pairwise using a BLAST search (Altschul et al. 1997).
Yeasts were identified by analysis of the D1/D2 region of the
large subunit of rRNA (LSU rRNA) gene sequence similarities
(Kurtzman & Robnett 1998). Methods for DNA extraction and
amplification of the D1/D2 domain were described previously
(Limtong et al. 2007). The D1/D2 domain of the LSU rRNA gene
was PCR amplified from yeast genomic DNA. The D1/D2 region
of the LSU rRNA gene was amplified and sequenced with
primers, NL1 and NL4 (Kurtzman & Robnett 1998). The PCR
products were checked by agarose gel electrophoresis and purified using HiYieldä Gel/PCR DNA Fragments Extraction Kit
(RBC Bioscience, Chung Ho City, Taipei, Taiwan). The purified
products were submitted to Macrogen (Korea) for sequencing.
Sequencing reactions were performed in a MJ Research PTC225 Peltier Thermal Cycler, using an ABI PRISMÒ BigDyeä Terminator Cycle Sequencing Kit with AmpliTaqÒ DNA polymerase (FS enzyme) (Applied Biosystems, USA), following the
protocols supplied by the manufacturer. Single-pass sequencing was performed on each template using the primers NL1
and NL4 for the D1/D2 region of the LSU rRNA gene. The
fluorescent-labelled fragments were purified from the unincorporated terminators with an ethanol precipitation protocol. The samples were resuspended in distilled water and
subjected to electrophoresis in an ABI 3730XL sequencer (Applied Biosystems, USA). The sequences were compared pairwise using a BLAST search (Altschul et al. 1997).
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