MIT Biology Department
7.012: Introductory Biology - Fall 2004
Instructors: Professor Eric Lander, Professor Robert A. Weinberg, Dr. Claudette Gardel
Solutions to 7.012 Recombinant DNA/Cloning
a) Plasmid p7013 would give a single band at 3000 bp and the tet gene fragment would
give a single band at 200 bp.
b) When you mix the DNA and bacteria in the transformation protocol, most of the bacteria in
the transformation reaction do not take up a plasmid. The presence of ampicillin prevents all
these other bacteria from growing and allows you to select only those containing the
ampicillin resistance gene.
c)
EcoRI
StuI
r
tet gene
SalI
PvuII
EcoRI
pp7012
7013
ori
r
amp gene
SalI EcoRIStuI
SalI
p7013-AT-1
ori
Plasmid 1
r
amp gene
Plasmid 2
r
tet gene
SalI
EcoRI
PvuII
EcoRI
StuI
SalI
p7013-AT-2
ori
r
amp gene
Plasmid 3
d) A selection for tetracycline and ampicillin resistance would produce only plasmids 2 and 3,
molecules that contain both the genes for ampicillin and tetracycline resistance.
e) Use EcoRI and SalI to cut both the p7013 plasmid and the tet gene fragment. This procedure
generates two different "sticky" ends on each molecule, which will allow the tet gene to insert
in only one orientation.
You could also cut p7013 with StuI and SalI, and cut the tet gene fragment with PvuII
and SalI. StuI and PvuII both generate "blunt" ends that can be ligated together.