Bio-Rad Ligation and Transformation Module - Manual (Page 53)

By digesting a small portion of the miniprep DNA with Bgl II enzyme (see
instructions 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 (2,974 bp) and the other the size of the
inserted DNA (the original PCR product). 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.
Preparation for Restriction Digestion Analysis
1. Label one microcentrifuge tube with your initials and "Bgl II master
mix."
2. The restriction digestion reactions will be performed on each miniprep
in a final volume of 20 µl with 10 µl of plasmid DNA.
3. Prepare a master mix for Bgl II restriction enzyme digestion according
to the following table, using stock reagents from the common workstation.
Use a fresh tip for each reagent.
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