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

2. 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

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

5. Agarose gel analysis of PCR DNA

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

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

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

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

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