Computational protocol: Production and Partial Characterization of an Alkaline Xylanase from a Novel Fungus Cladosporium oxysporum

Similar protocols

Protocol publication

[…] Genomic DNA of fungal isolate was extracted from fresh cultures and used as template for PCR amplification using a method described previously []. The ITS1-5.8S-ITS2 rDNA region of the fungi was amplified by PCR using primer set pITS1 (5′-TCCGTAGGTGAACCTGCCG-3′) and pITS4 (5′-TCCTCCGCTT-ATTGATATGC-3′). PCR amplification conditions were as follows: predenaturing at 95°C for 5 min, 30 cycles of denaturation at 95°C for 1 min; annealing at 55°C for 1 min; and prolonging at 72°C for 1 min, before the extension at 72°C for 10 min and final cooling to 4°C. Electrophoresis was used to examine PCR products in a 0.8% (w/v) agarose gel in 1× TAE buffer (0.4 M Tris, 50 mM NaOAc, 10 mM EDTA, pH 7.8). The amplified DNA fragment was cloned into the pMD18-T vector (Takara, Japan) and its nucleotide sequence was determined using an Applied Biosystems 377B automatic DNA sequencer (Applied Biosystems, Foster, CA, USA). The resulting sequences obtained were queried against the GenBank database using Basic Local Alignment Search Tool (BLAST) program. The neighbor-joining (NJ) method and maximum parsimony (MP) method from the PHYLIP suit program were used for constructing the phylogenetic tree. Bootstrap analysis was used to evaluate the tree topology of the NJ data by performing 1,000 resamplings and marking the branching points. The evolutionary distance matrix was estimated according to Kimura's two-parameter model. […]

Pipeline specifications

Software tools BLASTN, PHYLIP
Application Phylogenetics
Organisms Triticum aestivum
Chemicals Nitrogen