C dna genomiclibrary3
-
Upload
bruno-thadeus -
Category
Technology
-
view
267 -
download
0
Transcript of C dna genomiclibrary3
![Page 1: C dna genomiclibrary3](https://reader033.fdocuments.us/reader033/viewer/2022060118/55897cffd8b42a7e758b45a7/html5/thumbnails/1.jpg)
![Page 2: C dna genomiclibrary3](https://reader033.fdocuments.us/reader033/viewer/2022060118/55897cffd8b42a7e758b45a7/html5/thumbnails/2.jpg)
![Page 3: C dna genomiclibrary3](https://reader033.fdocuments.us/reader033/viewer/2022060118/55897cffd8b42a7e758b45a7/html5/thumbnails/3.jpg)
Subculture colony producing insulin onto new agar plate
Oligo(dT)
primer
Fish pancreas
extract mRNA
AAAnA-3’5’AAAnA-3’5’mRNA
TTTn T-5’
poly(A) tail AAAnA-3’5’
TTTn T-5’3’Reverse transcript
ase
dNTPs
mRNA mRNA
cDNAOligo(dT)
primer
Alkali digestion of mRNA template/RNaseH
TTTnT-5’
3’
TTTn T-5’
AAAnA-3’
dsDNA with a ss hairpin susceptible
to S1 nuclease
cDNA
DNA polymera
se I
dNTPs
S1 nuclease
TTTn T-5’
AAAnA-3’
dsDNA
Clone fragments onto plasmid vector
Recombinant
plasmid
dsDNA
Plasmid Transform E. coli
Screen colony producing insulin by replica plating on nitrocellulose
membrane followed by
staining using radio-labelled
antibodies specific to insulin
Bacterial colonies on agar plate
Colony producing
insulin
Harvest insulin
![Page 4: C dna genomiclibrary3](https://reader033.fdocuments.us/reader033/viewer/2022060118/55897cffd8b42a7e758b45a7/html5/thumbnails/4.jpg)
![Page 5: C dna genomiclibrary3](https://reader033.fdocuments.us/reader033/viewer/2022060118/55897cffd8b42a7e758b45a7/html5/thumbnails/5.jpg)
![Page 6: C dna genomiclibrary3](https://reader033.fdocuments.us/reader033/viewer/2022060118/55897cffd8b42a7e758b45a7/html5/thumbnails/6.jpg)
![Page 7: C dna genomiclibrary3](https://reader033.fdocuments.us/reader033/viewer/2022060118/55897cffd8b42a7e758b45a7/html5/thumbnails/7.jpg)
![Page 8: C dna genomiclibrary3](https://reader033.fdocuments.us/reader033/viewer/2022060118/55897cffd8b42a7e758b45a7/html5/thumbnails/8.jpg)
![Page 9: C dna genomiclibrary3](https://reader033.fdocuments.us/reader033/viewer/2022060118/55897cffd8b42a7e758b45a7/html5/thumbnails/9.jpg)
DNA sequence analysisDNA sequence analysis
• The dideoxy nucleotide technique (Sanger)
![Page 10: C dna genomiclibrary3](https://reader033.fdocuments.us/reader033/viewer/2022060118/55897cffd8b42a7e758b45a7/html5/thumbnails/10.jpg)
DNA sequence analysisDNA sequence analysis