pKAN-R/pARA Sequence Biotechnology Lab Program Marty Ikkanda Bruce Wallace pKAN-R/pARA Sequence Laboratory Protocols by: Marty Ikkanda Powerpoint by: Anthony Daulo Pierce College, Woodland Hills, CA V.1.2.4

pKAN-R pARA Restriction analysis of pKAN-R and pARA rfp Bruce Wallace 5408 bp BamH I rfp 702 bp pARA 4058 bp Hind III BamH I Hind III 40 bp

Restriction fragments after digest with Hind III and BamH I Restriction analysis of pKAN-R and pARA Bruce Wallace Restriction fragments after digest with Hind III and BamH I BamH I Hind III 4018 bp BamH I Hind III 4706 bp Hind III BamH I 702bp Hind III BamH I 40 bp

Ligation of pKAN-R/pARA restriction fragments Bruce Wallace sticky end BamH I 3’ 5’ 3’ sticky end Hind III 5’ sticky end Hind III 5’ 3’ sticky end BamH I 5’ 5’ 3’ 3’ 3’ 5’

Recombinant plasmid of interest Ligation of pKAN-R/pARA restriction fragments Bruce Wallace Recombinant plasmid of interest pARA-R 4720 bp BamH I rfp 702bp Hind III

Preparing competent cells for transformation Bruce Wallace Lipid bilayer (inner) Adhesion zone Peptidoglycan layer Lipid bilayer (outer) Calcium ions

Transforming Escherichia coli with pARA-R Bruce Wallace Competent Cells pARA-R Recombinant Plasmids

Transforming Escherichia coli with pARA-R Bruce Wallace Lipid bilayer (inner) Adhesion zone Peptidoglycan layer Lipid bilayer (outer) Calcium ions pARA-R

Growth of transformed bacteria on various plates Bruce Wallace P+ plates LB LB/amp LB/amp/ara P- plates No growth LB LB/amp

Colony isolation and culture Preparing an overnight culture of E. Coli for RFP expression Bruce Wallace Colony isolation and culture LB/amp/ara broth