*This page is part of
undergraduate assignment for Molecular Biology at Davidson College
Cloning 5 Isoforms
of Isocitrate Dehydrogenase
By
Elizabeth Shafer
Digestion Experiment Design
Digestion of IDH1 plasmid:
Eco RI cuts the plasmid
construct 42 base pairs upstream of the insert. Eco R I cuts the IDH1 gene
resulting in a 390 bp fragment and 694 bp fragment. Thus a plasmid with an
insert with the correct orientation would drop a large band at the top
corresponding to the plasmid construct and a smaller band which is
approximately 432 base pairs long. A plasmid with the insert in the reverse
orientation would drop a same band corresponding to the plasmid construction
and a band which is 736 base pairs long.
Digestion of IDH2 plasmid:
Eco R V cuts IDH2 18 base
pairs upstream of the insert. Eco RV cuts the IDH2 gene resulting in a 790 bp
fragment and 320 bp fragment. Thus a plasmid with an insert with the correct
orientation would drop a large band at the top corresponding to the plasmid
construct and a smaller band which is approximately 808 base pairs long. A
plasmid with the insert in the reverse orientation would drop a same band
corresponding to the plasmid construction and a band which is 338 base pairs
long.
Digestion of IDP1plasmid:
Bgl II cuts IDP1 but does
not cut the plasmid. Therefore, the plasmid must be digested first with Eco RV
which does not cut IDP1 to excise the fragment. Bgl II cuts IDP1 resulting in a
1208 bp fragment and a79 bp fragment.
Thus a plasmid with an insert with the correct orientation would drop a
large band at the top corresponding to the plasmid construct and a smaller band
which is approximately 1226 base pairs long. A plasmid with the insert in the
reverse orientation would drop a same band corresponding to the plasmid
construction and a band which is 97 base pairs long.
Digestion of IDP2 plasmid:
Eco RI cuts the plasmid
construct 42 base pairs upstream of the insert. Eco RI cuts IDP2 resulting in a
348 bp fragment and 891 bp fragment. Thus a plasmid with an insert with the
correct orientation would drop a large band at the top corresponding to the
plasmid construct and a smaller band which is approximately 390 base pairs
long. A plasmid with the insert in the reverse orientation would drop a same
band corresponding to the plasmid construction and a band which is 933 base
pairs long.
Digestion of IDP3 plasmid:
Cla I cuts IDP3 but does not
cut the plasmids, therefore the plasmid must be digested first with Eco RV
which does not cut IDP3 to excise the fragment. Cla I cuts IDP3 resulting in a
495 bp fragment and 768 bp fragment.
Thus a plasmid with an insert with the correct orientation would drop a
large band at the top corresponding to the plasmid construct and a smaller band
which is approximately 513 base pairs long. A plasmid with the insert in the
reverse orientation would drop a same band corresponding to the plasmid
construction and a band which is 786 base pairs long.
Isocitrate
Dehydrogenase Main Page
Created by: Elizabeth Shafer. Email questions to lishafer@davidson.edu