Bio-Rad Ligation and Transformation Module - Manual (Page 52)

Appendix B
Restriction Digestion of Plasmid DNA with
Bgl II Enzyme
Instructors Advanced Preparation
The Ligation and Transformation module contains Bgl II enzyme and reaction
buffer to enable analysis of plasmids derived using the module and
subsequently purified using a plasmid purification protocol. Electrophoresis
reagents, including sample loading dye and a molecular weight ruler are
also required for the analysis. The pJet1.2 plasmid vector is 2,974 bp in
length, thus after restriction digestion of plasmids and subsequent
electrophoresis a band of around 3 kb and a smaller band corresponding
to the size of the PCR product insert is expected.
To digest and analyze four plasmids, each student team requires:
Item Qty
Microcentrifuge tubes 4–8
Bgl II restriction enzyme 4 µl
10x Bgl II reaction buffer 8 µl
Sterile water 30 µl
1% agarose gel 1
Electrophoresis running buffer (sufficient to fill
electrophoresis chamber)
Sample loading dye, 5x
50 µl
Molecular weight ruler (10 µl of EZ load molecular ruler per gel is
recommended)
2–20 µl adjustable volume pipet and tips
1
Background
Once transformed bacteria are grown overnight in a liquid culture (see
Appendix A), a miniprep is performed to purify the plasmid DNA from the
bacteria. Before proceeding with further experiments using the purified
plasmid DNA, it is important to verify that the isolated plasmid contains the
insert of interest. During the ligation stage, a gene of interest was
ligated into the pJet1.2 blunted vector, which contains a Bgl II restriction
enzyme recognition site on either side of the insertion site. To determine
the size of the fragment inserted, the purified plasmid can be analyzed by
restriction enzyme digestion with Bgl II enzyme and subsequent agarose
gel electrophoresis.
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