Bio-Rad Ligation and Transformation Module - Manual (Page 29)

Looking back at earlier steps in the experiment, a gene or portion of a
gene was ligated into the plasmid vector. From previous work, the size of
this insert should be known. By digesting a small portion of the miniprep
DNA with a specific restriction enzyme such as Bgl II (see below), the
insert should be cut out of the vector. Running the products of the restriction
digestion on an agarose gel should give two DNA bands, one the size of
the vector and the other the size of the inserted DNA.
If there are more than two bands in any digest, it may mean that the insert
contains a Bgl II site. Does the size of the excised bands add up to the
size of the original PCR fragment? Alternatively, two similarly sized fragments
may indicate that a mixed culture was used to start the miniprep, instead
of an isolated colony.
Restriction enzyme digestion analysis of plasmid DNA. Circular plasmid DNA purified from
bacterial minipreps is isolated and digested with Bgl II, a restriction enzyme producing at least
two linearlized fragments — vector DNA and PCR fragment (lane 2). These fragments can be
visualized using agarose gel electrophoresis. If the PCR fragment contains a Bgl II restriction
site, three DNA bands may be observed (lane 3). A 500 bp molecular weight ruler is shown in
lane 1.
BgI II
PCR
Fragment
PCR Fragment
BgI II
Bgl II
Digest
1 2 3
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