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Lots of tools for cloning:. Plasmid (BAC) Phagemid Cosmid Lambda (PAC). Needs to be at least 40kb to grow, but A left and right arm can be made smaller… See next slide. Intron and Exon in Eukaryotic Cells. exon. exon. exon. intron. intron. 5’. 3’. DNA. promotor. 3’. 5’.

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Presentation Transcript
slide1

Lots of tools for cloning:

Plasmid (BAC)

Phagemid

Cosmid

Lambda (PAC)

slide4

Needs to be at least 40kb to grow, but

A left and right arm can be made smaller…

See next slide

slide8

Intron and Exon in Eukaryotic Cells

exon

exon

exon

intron

intron

5’

3’

DNA

promotor

3’

5’

stop codon

start codon

Transcription

mRNA

Processing

cap

poly A

tail

Splicing

Intron deleted

mature mRNA

To cytoplasm

Take place in nucleus

eukaryotes splice signals

AG/GT

CAG/NT

exon 1 intron 1 exon 2

Eukaryotes Splice Signals
slide11

cDNA Is Reverse Transcribed from mRNA

5’

3’

3’

5’

5’

Baltimore, Dulbecco, Temin (1975)

mature mRNA

poly A tail

Reverse

transcription

TTTT

3’

5’

RNA hydrolysis

DNA polymerase

3’

CCC

5’

GGG

3’

slide12

cDNA Library

Genes in expression

mRNA

Reverse transcription

Complete

gene

cDNA

Vector: Plasmid

slide13

Libraries: cDNA

cDNA library has sequences only from genes transcribed into mRNA (~3% of genome)

slide17

MNGTEGPNFYVPFSNKTGVVRSPFEAPQYYLAEPWQFSMLAAYMFLLIML

GFPINFLTLYVTVQHKKLRTPLNYILLNLAVADLFMVFGGFTTTLYTSLH

GYFVFGPTGCNLEGFFATLGGEIALWSLVVLAIERYVVVCKPMSNFRFGE

NHAIMGVAFTWVMALACAAPPLVGWSRYIPEGMQCSCGIDYYTPHEETNN

ESFVIYMFVVHFIIPLIVIFFCYGQLVFTVKEAAAQQQESATTQKAEKEV

TRMVIIMVIAFLICWLPYAGVAFYIFTHQGSDFGPIFMTIPAFFAKTSAV

YNPVIYIMMNKQFRNCMVTTLCCGKNPLGDDEASTTVSKTETSQVAPA*

slide18

Maxim-Gilbert

Sequencing

MNGTEGPNFYVPFSNKTGVVRSPFEAPQYYLAEPWQFSMLAAYMFLLIML

GFPINFLTLYVTVQHKKLRTPLNYILLNLAVADLFMVFGGFTTTLYTSLH

GYFVFGPTGCNLEGFFATLGGEIALWSLVVLAIERYVVVCKPMSNFRFGE

NHAIMGVAFTWVMALACAAPPLVGWSRYIPEGMQCSCGIDYYTPHEETNN

ESFVIYMFVVHFIIPLIVIFFCYGQLVFTVKEAAAQQQESATTQKAEKEV

TRMVIIMVIAFLICWLPYAGVAFYIFTHQGSDFGPIFMTIPAFFAKTSAV

YNPVIYIMMNKQFRNCMVTTLCCGKNPLGDDEASTTVSKTETSQVAPA*

slide34

Logo for

a) Splice acceptor

b) Splice Donor

c) Initiator Met

slide35

AG/GT

CAG/NT

exon 1 intron 1 exon 2