PCR primer design ATTGCCACGTTGACGGTCAG CACGATTTCCGGAAGTAC Inducer IPTG lactose

PCR: primer design ATTGCCACGTTGACGGTCAG CACGATTTCCGGAAGTAC

Inducer: IPTG, lactose

Restriction endonucleases • Palindromes • Blunt ends v. s. sticky ends

重組蛋白質 在寄主(host)細胞中的表達 基因 優點 • 產量大 • 操作易 質體 染色體 4, 000 kb E. coli 缺點 • no post-translation modification • inclusion body • not secrete protein

重組蛋白質 在寄主細胞中的表達 E. coli 缺點 not secrete protein gram-positive bacteria B. subtilis proteases limited vector & promoter “signal” has not found yet

Growth inhibition

常見的動物細胞寄主 insect cell-baculovirus mammal-retrovirus chinese hamster ovary (CHO) cells immortal cell

Baculovirus Replication and Gene Expression Infection Gene Expression and Protein Synthesis: • Immediately-Early Genes • Delay-Early Genes • Late Genes • Very Late Genes: polyhedrin and p 10 Virus Assembly and Release

Infect cultured insect cells Cotransfection with Ac. MNPV DNA Amplify and titer the recombinant virus Confirm presence of target gene by PCR or Southern blot Plaque Assay for putative recombinant virus

Generation of Recombinant Baculovirus by Bacmid

Molecular tools • • Gel Electrophoresis Labeled Tracers Hybridization Mapping and quantifying transcripts Measuring transcription rates in vivo Assaying DNA-protein interactions Knockouts

Pulse-field gel electrophoresis (0. 2 -2. 2 Mb)

Two-dimensional gel electrophoresis

Ion exchange chromatography R+ A- + B - R+ B - + A -

Gel filtration chromatography

Affinity chromatography

Molecular tools • • Gel Electrophoresis Labeled Tracers Hybridization Mapping and quantifying transcripts Measuring transcription rates in vivo Assaying DNA-protein interactions Knockouts

Molecular tools • • Gel Electrophoresis Labeled Tracers Hybridization Mapping and quantifying transcripts Measuring transcription rates in vivo Assaying DNA-protein interactions Knockouts

DNA fingerprinting

The Sanger dideoxy method of DNA sequencing Figure 5. 18

Molecular tools • • Gel Electrophoresis Labeled Tracers Hybridization Mapping and quantifying transcripts Measuring transcription rates in vivo Assaying DNA-protein interactions Knockouts

S 1 mapping the 5’-end of a transcript

Primer extension

Molecular tools • • Gel Electrophoresis Labeled Tracers Hybridization Mapping and quantifying transcripts Measuring transcription rates in vivo Assaying DNA-protein interactions Knockouts

Molecular tools • • Gel Electrophoresis Labeled Tracers Hybridization Mapping and quantifying transcripts Measuring transcription rates in vivo Assaying DNA-protein interactions Knockouts

Figure 5. 35

DNase footprinting Page 125

Molecular tools • • Gel Electrophoresis Labeled Tracers Hybridization Mapping and quantifying transcripts Measuring transcription rates in vivo Assaying DNA-protein interactions Knockouts