Overview of Recombinant DNA Techniques

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Presentation transcript:

Overview of Recombinant DNA Techniques Dr. Fu-Pang Lin

pETBlchiI construction pETBlchiI target gene BamHI Specific N-ter primer pETBlchiI Xho I Specific C-ter primer target gene BamHI Xho I His tag MCS T7 terminator T7 promoter BamHI Target gene pET20b(+) Lac I T7 promoter Xho I His tag Amp Recombinant plasmid Ori Lac I Amp pETBlchiI construction Ori

Template DNA preparation primers design PCR amplification and DNA Cloning Recombinant DNA analysis Gene expression Recombinant Protein purification Recombinant Protein biocharacterization

Agarose gel analysis of PCR DNA

Fast screen of recombinant plasmid clones ★ ★ ★ ★ ★ ★ ★ ★

Lysate PCR of recombinant plasmid clones T7 p / T7 t T7 p / T7 t GSP GSP

PCR and RE digest confirmation of the recombinant clone plasmid T7 p / T7 t GSP RE digest

SDS-PAGE analysis of the recombinant enzyme (CBR protein staining)

Activity staining of the purified recombinant enzyme A. CBR staining B. Activity staining

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