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Overview of Recombinant DNA Techniques

Overview of Recombinant DNA Techniques. Dr. Fu-Pang Lin. BamHI. Specific N-ter primer. pET BlchiI. Xho I. Specific C-ter primer. target gene. BamHI. His tag. Xho I. MCS. T7 terminator. T7 promoter. BamH I. Target gene. pET20b(+). Lac I. T7 promoter. Xho I. His tag. Amp.

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Overview of Recombinant DNA Techniques

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  1. Overview of Recombinant DNA Techniques Dr. Fu-Pang Lin

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

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

  4. Agarose gel analysis of PCR DNA

  5. Fast screen of recombinant plasmid clones ★ ★ ★ ★ ★ ★ ★ ★

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

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

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

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

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