Cloning DNA

1. Cloning DNA May 4

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

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 G CTTAA 5’ AATTC G 3’ 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 5’ PvuII

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 G CCTAG GGATCC CCTAGG 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 M GATC CTAG Dam AlwI GGATC TGATCA BclI M BspDI ATCGATc XbaI TCTAGAtc M CCAGG GGTCC GGTACCagg Acc65I Dcm M

11. mcr and mrr systems

12. joining DNA fragments • ligase • topoisomerase

13. Invitrogen

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

15. E. coli strains