Molecular Cloning Techniques for Applications in Biotechnology

This project focuses on cloning the cel48 glycoside hydrolase gene, a cellulase involved in cellulose degradation, using foundational molecular biology techniques. DNA samples were quantified and assessed for purity, then amplified using PCR with gene-specific primers. Amplification was confirmed through agarose gel electrophoresis, followed by extraction and purification of the target DNA fragment.

The purified insert was ligated into the pGEM-T Easy vector and transformed into E. coli TOP10 competent cells to generate recombinant clones. Successful transformants were identified by blue-white screening and subjected to downstream plasmid extraction and Sanger sequencing. Sequence alignment revealed several point mutations relative to the reference cel48 gene, highlighting common challenges in PCR-based cloning, including polymerase errors and template quality effects.

Overall, the project demonstrates the complete workflow of classical gene cloning, from amplification and purification to ligation, transformation, and sequence validation, while also underscoring the importance of quality control, optimization, and careful data interpretation in molecular cloning experiments.

Wet Lab Methods:

• DNA quantification using NanoDrop spectrophotometry
• PCR amplification with gene-specific primers
• Agarose gel electrophoresis for size verification
• Gel extraction and purification of the amplified fragment
• Ligation into the pGEM-T Easy vector
• Transformation into E. coli TOP10 competent cells
• Blue-white screening for recombinant identification
• Plasmid isolation (miniprep) and Sanger sequencing
• Sequence alignment and mutation analysis
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Software / Tools

• Benchling (sequence visualization and primer analysis)
• BLAST for sequence identity checks
• SnapGene or equivalent DNA analysis tools
• Excel / Google Sheets for quantification and result tracking