Restriction mapping sample question

You have a 5.3 kb PstI fragment cloned into the PstI site of the vector pUC19, which is 2.7 kb in size. This vector has unique sites for the following enzymes in a multiple cloning site:

PstI, HincII, XbaI, BamHI, SmaI, EcoRI

A restriction map of the 5.3 kb insert is prepared. The recombinant plasmid is digested with the enzymes listed above in single digests, and then several combinations of enzymes are tested in double digests. The following bands are observed when the digests are run on a gel:

Enzyme(s) used	Band sizes observed (kb)
PstI	5.3, 2.7
EcoRI	5.4, 2.6
HincII	4.5, 3.5
SmaI	6.7, 1.3
XbaI	4.0 (high intensity band)
BamHI	3.9, 3.7, 0.4
HincII + XbaI	4.0, 3.5, 0.5
HincII + EcoRI	3.5, 2.6, 1.9
XbaI + BamHI	3.7, 3.6, 0.4, 0.3
EcoRI + BamHI	3.7, 2.2, 1.7, 0.4
SmaI + BamHI	3.7, 3.0, 0.9, 0.4
HincII + BamHI	3.9, 3.5, 0.4, 0.2

Use the data above to construct a map of the cloned insert. Note that fragments smaller than 100 bp will not usually be visible on a gel, and that 2 fragments with a similar size will run at the same spot on a gel – resulting in a brighter band.