To clone the selected genes for expression, the genomic DNA was isolated from the Leptospira interrogans, serovar Copenhageni strain Fiocruz L1-130 using the genomic DNA isolation kit, Himedia. The gene sequences corresponding to the proteins, SP, Hydrolase (HYD) and Len A were amplified from the genomic DNA using the specific primers (HYD-F 5′-AAAGATCTATGAGATCGGAAAGAATTGC-3′, HYD-R 5′-AACTCGAGTTAGATTTGAGAAGAATGCTC-3′, Len A-F 5′-AAAAGCTTGCATGAATTT AAAACAAG G-3′, Len A-R 5′-AACTCGAGTTACTGTTCTACACAGAGAAG-3′, SP-F 5′-AAGGATCCA TGAGTAGAAGTTC TTCCAACC-3′, SP-R 5′- AAGCGGCCGCTTAGAACGCTTTTCCTAATATG-3′) and cloned in pET-28a (+) expression vector (Novagen-Cat. # 69864-3). The clones were confirmed by sequencing.
Genomic dna isolation kit
Manufactured by HiMedia
Sourced in India
The Genomic DNA isolation kit is a laboratory equipment designed to extract and purify genomic DNA from various biological samples. It utilizes a standardized protocol to efficiently isolate high-quality DNA for downstream applications such as PCR, sequencing, and molecular analysis.
Automatically generated - may contain errors
Lab products found in correlation
Chitin beads, by New England Biolabs (1 mentions)
Terminal transferase, by Roche (1 mentions)
Nbt bcip solution, by Roche (1 mentions)
Anti dig antibody, by Roche (1 mentions)
Ni nta matrix, by Qiagen (1 mentions)
E coli topoisomerase 1, by New England Biolabs (1 mentions)
Dig labeled ddutp, by Roche (1 mentions)
Lysozyme, by Merck Group (1 mentions)
Taq dna polymerase master mix red, by Ampliqon (1 mentions)
4 protocols using genomic dna isolation kit
1
Cloning of Leptospira Virulence Genes
2
Biochemical Toolkit for DNA Manipulation
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Restriction enzymes, vectors (pTWIN1 & pMBX10), DNA (ØX virion, ØX dsDNA and M13mp18 dsDNA), E. coli Topoisomerase I and chitin beads were obtained from New England Biolabs, UK. Q-Sepharose, Sephadex 50 columns, terminal transferase, Dig labeled ddUTP, anti-Dig antibody and NBT-BCIP solution were obtained from Roche Life Sciences, UK. Ni–NTA matrix was obtained from Qiagen, Germany. IPTG, ATP, phosphocreatine, phosphocreatine kinase and E. coli Ssb protein were obtained from Sigma–Aldrich India. Bacterial growth medium component were obtained from BD and Co., India. Oligo dT50 was obtained from MWG Biotech, India. Genomic DNA isolation kit was obtained from Hi-media Laboratories, India.
3
Genomic DNA Extraction Protocol
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The media, chemicals and genomic DNA isolation kit used in this study were purchased from HiMedia, Mumbai, India. Lysozyme and proteinase were obtained from Sigma-Aldrich, USA. Taq DNA Polymerase Master Mix Red was procured from Ampliqon, Denmark. All chemicals used in this study were of analytical grade.
4
Bacterial Genomic DNA Isolation and 16S rRNA Sequencing
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Chromogenic bacterial strain BRT-GR3 was subjected to genomic DNA isolation using genomic DNA isolation kit procured from Himedia. To 5 µl of isolated genome 25 μL of PCR reaction solution (1.5 μL of Forward Primer (8F-AGAGTTTGATCCTGG CTCAG) and Reverse Primer (1541R-AAGGAGGTGATCCAGCCGCA)), 5 μL of deionized water, and 12 μL of Taq Master Mix) were added and PCR were set up with respective conditions. The PCR products were purified and subjected to sequencing. Singlepass sequencing was performed on each template using below 16s rRNA universal primers. The fluorescent-labeled fragments were purified from the unincorporated terminators with an ethanol precipitation protocol. The samples were resuspended in distilled water and subjected to electrophoresis. The sequence was refined manually after crosschecking with the raw data to remove ambiguities and was submitted to the Gene Bank and accession number was obtained.
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