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The resultant product was gene-cleaned using Nucleogen gene clean kit, ligated in T-vector using Takara Perfect T-cloning kit, and then inserted into DHa E. coli mutant cells (RBC) by overnight incubation (37 C). Transformed cells were selected, grown overnight (37 C) in LB broth and their plasmids were extracted using SolGent Plasmid mini-prep kit, and were later sequenced. The BLAST search program was used to look for nucleotide sequence homology for the 18S ITS (1/4) region for fungi. The sequences obtained were aligned by Clustal W using MEGA version 4 software , and the neighbor-joining tree was generated using same software. Bootstrap replications (1 K) were used for a statistical support for the nodes in the phylogenetic tree. |
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