Cloning DNA May 4.

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Presentation transcript:

Cloning DNA May 4

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

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

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’

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

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

Cleavage close to the ends NEB reference

Star (*) activity

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

mcr and mrr systems

joining DNA fragments ligase topoisomerase

Invitrogen

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

E. coli strains

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