Bio-Rad Ligation and Transformation Module - Manual (Page 54)

Reagent Volume for 1 Reaction Volume for 5 Reactions
10x Bgl II reaction buffer 2 µl 10 µl
Sterile water 7 µl 35 µl
Bgl II restriction enzyme 1 µl 5 µl
Total 10 µl 50 µl
Experimental Procedure for Restriction Digestion Analysis
1. Label a microcentrifuge tube for each plasmid miniprep.
2. Prepare digestion reactions by combining 10 µl of the Bgl II master mix
and 10 µl of each plasmid DNA in the appropriately labeled
microcentrifuge tubes.
3. Mix tube components and spin briefly in a microcentrifuge to collect the
contents at the bottom of the tube.
4. Incubate reactions at 37°C for 1 hr. If the reactions will not be analyzed
by agarose gel electrophoresis immediately, store them at –20°C until
analysis.
Preparation for Electrophoresis
1. Place a 1% agarose gel in the electrophoresis chamber and add
electrophoresis running buffer to just cover the gel.
2. Plan your gel electrophoresis experiment.
3. Add the appropriate amount of sample loading dye to the DNA samples
and pipet up and down to mix.
4. Load 20 µl of the restriction digestion reactions on the gel according to
your plan and load an appropriate amount of molecular weight ruler in
a separate lane.
5. (Optional) It is recommended that undigested DNA (at the same dilution
as your restriction digests) also be run next to your digested samples.
6. Electrophorese samples at 100 V for 30 min and analyze results.
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