Restriction mapping techniques

– usual technique

– do single & double digests with various enzymes

– add up fragment sizes to fit with each other & vector

– like sample question

– alternate technique

– do partial digests on DNA radiolabelled at one end

– RE digest plasmid with cloned insert

– use 2 REs found in multiple cloning site near insert

– 1 RE produces 5' ends, 1 produces 3' ends

– Klenow + radiolabelled dNTP will end-fill 5' end

– 3' end not labelled

– all linearized plasmids labelled at one end

– do partial digest with 3^rd RE – run products on gel

– only fragments visible on autoradiograph

– end-labelled ones – all have 1 end of insert

– different- sized fragments

– show order of distances between sites