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Cloning DNA May 4.

Published byVictor Sherman Modified over 8 years ago

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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

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