Protocol: Size Determination of Plasmid DNA Using Agarose Gel Electrophoresis

OVERVIEW

Plasmids are circular DNA. Their size can not be determined in their native state as the migration of circular DNA in agarose gel is quite different and can not be compared with the linear fragments of ladder DNA. To know the approximate size of plasmid, the first step is to linearize the plasmid using a restriction enzyme that generates strictly a single cut. This results in a linear DNA that can be run on agarose gel and its size can be compared with the ladder sequence.

REQUIREMENTS

Reagents and solutions
Electrophoresis buffer (TAE or TBE)
Plasmid DNA
Gel Loading dye
Agarose (standard)
DNA ladder
Appropriate restriction enzyme and buffer

Equipment and disposables
Set-up for agarose gel electrophoresis
Gel documentation system
Micropipettes and micropipette tips
Conical flask

PROCEDURE

Step 1: Digest the plasmid DNA with restriction enzyme
Note: Plasmid DNA must have only single site of the restriction enzyme

Step 2: Cast a 0.8 – 1% agarose gel
◊ Cast the agarose gels as described in section “Preparation of Agarose Gel for DNA Analysis”
◊ Choose the right concentration (percent) of agarose gel, depending on expected DNA fragments in the samples.

Step 3: When the gel is ready, place it in the electrophoresis chamber, fill with an electrophoresis buffer and load the samples (digested plasmid) and DNA ladder and start electrophoresis.

Follow the instructions given in the protocols “Running DNA Samples in Agarose Gel”

Step 4: When the electrophoresis is over, analyze the gel in the gel documentation system/uv-transilluminator.
Compare the location of the DNA band appearing in digested plasmid DNA sample with the known-size DNA bands of the DNA ladder.