Ro_Lecture13

Ro_Lecture13 - Plasmids Small circular autonomously...

Info iconThis preview shows pages 1–10. Sign up to view the full content.

View Full Document Right Arrow Icon
1 Plasmids Small circular autonomously replicating extrachromosomal DNA Bacterial genome (5000kb) Plasmid DNA (3kb) Modified plasmids, called cloning vectors Are used by molecular biologists to isolate Large quantities of a given DNA fragment Plasmids used for cloning share three properties Unique restriction site Antibiotic resistance Origin of replication
Background image of page 1

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
2 Plasmid elements Origin of replication: This is a DNA element that allows the plasmid to be replicated and duplicated in bacteria. Each time the bacterium divides, the plasmid also needs to divide and go with the daughter cells. If a plasmid cannot replicate in bacteria, then it will be lost.
Background image of page 2
3 Plasmid elements Antibiotic resistance: This allows for the presence of the plasmid to be selectively maintained in a given strain of bacteria Lab bacterial strains are sensitive to antibiotics. When grown on plates with antibiotics, they die. The presence of a plasmid with the antibiotics resistance gene allows these lab strains to grow on plates with the antibiotic. You are therefore selecting for bacterial colonies with the Plasmid
Background image of page 3

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
4 Plasmid elements Unique restriction sites: For cloning the plasmid needs too be linearized. Most cloning vectors have unique restriction sites. If the plasmid contains more than one site for a given restriction enzyme, this results in fragmentation of the plasmid Why does this matter?
Background image of page 4
5 pUC18 pUC18 is a commonly used plasmid: pUC= plasmid University of California Plasmid replicon copy No pBR322 pMB1 15 pUC18 pMB1 500 pACYC p15A 10 pSC101 pSC101 5
Background image of page 5

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
6 Why are plasmids important? Most genes are present as two copies in the entire genome. Plasmids allow us to obtain 1000’s of copies of a gene in a pure form
Background image of page 6
7 How is a specific gene isolated (CLONED)? Its like going to the library and looking for a specific book. It involves screening through a genomic library. A genomic library is a large collection of plasmids containing pieces of DNA from a specific species. The set of cloned fragments is so comprehensive that virtually the entire genome is represented in the library. The fragments that make up the library are initially generated by digesting genomic DNA (e.g. human) with a restriction enzyme- say EcoRI The EcoRI sites are randomly distributed in the genome- fragments of varying lengths will be generated. Some fragments will contain one gene, others two genes or cut genes in half. Gene1 Gene2 Gene3 A B C D E F
Background image of page 7

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
8 The library is random! Each fragment is cloned into the plasmid, each plasmid is put (transformed) into E.coli Gene1 Gene2 Gene3 A B C D E F A B C D
Background image of page 8
9 Fragments,bookmark title The library is not bookmarked or even titled and is in fragments! There is no organization to the library. It is simply a populations of cloned fragments representing the entire genome. The equivalent of this would be if you went to the University
Background image of page 9

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Image of page 10
This is the end of the preview. Sign up to access the rest of the document.

This note was uploaded on 04/15/2008 for the course BIO 105 taught by Professor Sullivan during the Fall '08 term at UCSC.

Page1 / 46

Ro_Lecture13 - Plasmids Small circular autonomously...

This preview shows document pages 1 - 10. Sign up to view the full document.

View Full Document Right Arrow Icon
Ask a homework question - tutors are online