Presentation is loading. Please wait.

Presentation is loading. Please wait.

Cloning DNA May 4.

Published byVictor Sherman Modified over 9 years ago

Similar presentations

Presentation on theme: "Cloning DNA May 4."— Presentation transcript:

1 Cloning DNA May 4

2 Cloning DNA the components host vector (plasmids, bacteriophage)
cutters (restriction endonucleases) joiners (ligase, topoisomerase) insert (DNA, mRNA->cDNA) host strains of E.coli

3

4 Restriction endonucleases
REs and methylases Type I, II, IIs, III enzymes sites and ends isochizomers compatible ends cleavage close to ends of linear DNA star activity heat inactivation methylases mcr and mrr restriction systems

5 Overhangs and blunt ends
3’ G CTTAA 5’ AATTC G 5’ GAATTC CTTAAG EcoRI 5’ 3’ 5’ CTGCA G 3’ G ACGTC 5’ CTGCAG GACGTC PstI 3’ 5’ CAG GTC CTG GAC 5’ CAGCTG GTCGAC PvuII 5’

6 Large and small sites GATC CTAG 4 bases MboI GGATCC CCTAGG 6 bases
BamHI 8 bases GCGGCCGC CGCCGGCG NotI

7 Compatible ends GATC CTAG GATC MboI CTAG GATCC G GGATCC CCTAGG G CCTAG
BamHI AGATCT TCTAGA GATCT A A TCTAG BglII

8 Cleavage close to the ends
NEB reference

9 Star (*) activity

10 Dam and Dcm methylases RE sites effected by methylases GATC CTAG Dam
AlwI GGATC BclI TGATCA M BspDI ATCGATc XbaI TCTAGAtc M CCAGG GGTCC GGTACCagg Dcm Acc65I M

11 mcr and mrr systems

12 joining DNA fragments ligase topoisomerase

13 Invitrogen

14 The inserts DNA RNA restriction digests of known sequences
shot-gun cloning PCR products forced cloning constructing libraries RNA reverse transcription

15 E. coli strains

Download ppt "Cloning DNA May 4."

Similar presentations

James Chappell & Cheuk Ka Tong

James Chappell & Cheuk Ka Tong
DNA Manipulation in Synthetic Biology. DNA structure Covalent bondsHydrogen bonds.

DNA Manipulation in Synthetic Biology. DNA structure Covalent bondsHydrogen bonds.
Restriction Enzymes. Restriction Endonucleases Also called restriction enzymes 1962: “molecular scissors” discovered in in bacteria E. coli bacteria have.

Restriction Enzymes. Restriction Endonucleases Also called restriction enzymes 1962: “molecular scissors” discovered in in bacteria E. coli bacteria have.
UNIT 2 MANIPULATION OF DNA AND GENE ISOLATION LECTURES: 9. DNA Cloning and Library Construction 10. Isolating Genes.

UNIT 2 MANIPULATION OF DNA AND GENE ISOLATION LECTURES: 9. DNA Cloning and Library Construction 10. Isolating Genes.
Restriction Enzymes Lecture 15: 1 11/20/2006 1. Definition: enzymes that recognize specific double-stranded sequences and hydrolyze the phosphodiester.

Restriction Enzymes Lecture 15: 1 11/20/ Definition: enzymes that recognize specific double-stranded sequences and hydrolyze the phosphodiester.
Restriction Enzymes.

Restriction Enzymes.
Recombinant DNA Isolation / Digestion / Fractionation Purification of the target fragment Cloning into vectors / Ligation Transformation of host cell /

Recombinant DNA Isolation / Digestion / Fractionation Purification of the target fragment Cloning into vectors / Ligation Transformation of host cell /
Restriction Analysis of Plasmid DNA

Restriction Analysis of Plasmid DNA
Restriction Enzyme Digestion of DNA. Experiment Goals Digestion of DNA by restriction enzyme Analyze digested DNA by electrophoresis.

Restriction Enzyme Digestion of DNA. Experiment Goals Digestion of DNA by restriction enzyme Analyze digested DNA by electrophoresis.
RFLP (Restriction Fragment Length Polymorphism). RFLP RFLP was developed at the late 70’s due to the discovery of restriction enzymes (REs; or called.

RFLP (Restriction Fragment Length Polymorphism). RFLP RFLP was developed at the late 70’s due to the discovery of restriction enzymes (REs; or called.
Biotechnology and Genetic Engineering PBIO 4500/5500 Eukaryotic gene organization Restriction enzymes Cloning vectors.

Biotechnology and Genetic Engineering PBIO 4500/5500 Eukaryotic gene organization Restriction enzymes Cloning vectors.
Enzymes used in Gene Manipulation

Enzymes used in Gene Manipulation
Restriction Enzymes. Molecular Scissors Restriction enzymes are like molecular scissors. They scan the DNA strand looking for particular sequences of.

Restriction Enzymes. Molecular Scissors Restriction enzymes are like molecular scissors. They scan the DNA strand looking for particular sequences of.
Molecular Biology Working with DNA. Topics  Genomic vs. Vector DNA  Purifying plasmid DNA  Restriction enzymes  Restriction maps.

Molecular Biology Working with DNA. Topics  Genomic vs. Vector DNA  Purifying plasmid DNA  Restriction enzymes  Restriction maps.
DNA aSsEmBlY tEcHnIqUes Design and construct novel biological organisms programmed by genetic circuits using standardized biological parts called BioBricks.

DNA aSsEmBlY tEcHnIqUes Design and construct novel biological organisms programmed by genetic circuits using standardized biological parts called BioBricks.
Restriction Enzymes DNA is a long chain of nucleotides. AluI recognises AGCT, and fixes itself on its target sequence. It cuts after the G to give Blunt.

Restriction Enzymes DNA is a long chain of nucleotides. AluI recognises AGCT, and fixes itself on its target sequence. It cuts after the G to give Blunt.
Recombinant DNA Introduction to Recombinant DNA technology

Recombinant DNA Introduction to Recombinant DNA technology
Recombinant DNA Technology. rDNA Technology Restriction Enzymes and DNA Ligase Plasmid Cloning Vectors Transformation of Bacteria Blotting Techniques.

Recombinant DNA Technology. rDNA Technology Restriction Enzymes and DNA Ligase Plasmid Cloning Vectors Transformation of Bacteria Blotting Techniques.
Restriction Mapping.

Restriction Mapping.
Restriction Endonucleases BIO450. Restriction Enzymes Enzymatic Activity Biological Role Diversity Recognition Sequence Digestion Conditions Typical Reaction.

Restriction Endonucleases BIO450. Restriction Enzymes Enzymatic Activity Biological Role Diversity Recognition Sequence Digestion Conditions Typical Reaction.

Similar presentations

Ads by Google