The target DNA fragments were cut from the agarose gel and purified using the TIAN gel Midi Purification Kit (TIANGEN BIOTECH, Beijing, China). Purified regenerants were then ligated into the pEASY-Blunt Simple Cloning Kit (TransGen Biotech, Beijing, China) and transformed into Escherichia coli Trans1-T1 Phage Resistant Chemically Competent Cells (TransGen Biotech, Beijing, China). The transformed cells were tiled on ampicillin LB medium containing IPTG and X-Gal for culturing at 37°C for 12–16 h. The positive clones were amplified to test for the target fragment, which was then sent to Takara Bio (Shanghai, China) for sequencing. DNAMAN version 6.0 software (Lynnon Biosoft, San Ramon, United States) and BioEdit version 7.1.3 software were used for assembling and aligning the gene sequences.
Escherichia coli trans1 t1 phage resistant chemically competent cells
Manufactured by Transgene
Sourced in China
Escherichia coli Trans1-T1 Phage Resistant Chemically Competent Cells are a genetically engineered strain of E. coli bacteria designed to be resistant to T1 phage infection. They are chemically treated to enhance their ability to take up and maintain foreign DNA during transformation experiments.
Automatically generated - may contain errors
2 protocols using escherichia coli trans1 t1 phage resistant chemically competent cells
1
DNA Fragment Cloning and Sequencing
2
Genomic Library Construction of A. solani
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The isolate used to construct the A. solani genomic library was provided by Professor Qinghe Chen of Fujian Academy of Agricultural Sciences, Fuzhou, China. The isolate was grown in PDA medium at 25°C for 2 weeks. Fungal mycelia were harvested and genomic DNA was extracted using plant gDNA kit (Promega) according to the manufacturer's instructions.
Genomic DNA (1 lg) was digested with HaeIII and DNA fragments were ligated with a linker prepared by mixing oligonucleotides A (5 0 -CTCTTGCTTGAATTCGGACTA-3 0 ) and B (5 0 -TAGTCCGAATTCAAGCAAGAGCACA-3 0 ) in an equal proportions. Ligated DNA fragments were amplified by PCR using oligonucleotide A as the primer (Karan et al., 2012) . The PCR products were purified using TIANgel Midi Purification kit (TianGen Biotech Ltd) and inserted into pMD19-T vector (TaKaRa). The pMD19-T gDNA was transformed into Escherichia coli Trans1-T1 phage-resistant chemically competent cells (Transgen Biotech Co. Ltd).
Genomic DNA (1 lg) was digested with HaeIII and DNA fragments were ligated with a linker prepared by mixing oligonucleotides A (5 0 -CTCTTGCTTGAATTCGGACTA-3 0 ) and B (5 0 -TAGTCCGAATTCAAGCAAGAGCACA-3 0 ) in an equal proportions. Ligated DNA fragments were amplified by PCR using oligonucleotide A as the primer (Karan et al., 2012) . The PCR products were purified using TIANgel Midi Purification kit (TianGen Biotech Ltd) and inserted into pMD19-T vector (TaKaRa). The pMD19-T gDNA was transformed into Escherichia coli Trans1-T1 phage-resistant chemically competent cells (Transgen Biotech Co. Ltd).
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