Protocol: Size Determination of DNA fragment Using Agarose Gel Electrophoresis

OVERVIEW

Approximate size of DNA fragments can easily be determined by agarose gel electrophoresis. This analysis is quite useful to determine if a DNA sample contains expected size DNA fragment(s) or not. To determine the size, the sample and DNA ladder (size marker), both are run in neighboring adjacent wells in an agarose gel. After the run is complete and appropriate separation is achieved among DNA fragments, the gel is analyzed by a gel documentation system to compare the location of unknown DNA fragments in DNA samples with the known size of DNA fragments of the ladder  to estimate the approximate size of unknown DNA fragments.

REQUIREMENTS

Reagents and solutions
Electrophoresis buffer (TAE or TBE)
DNA sample(s)
Gel Loading dye
Agarose (standard)
DNA ladder

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

PROCEDURE

Step 1: Cast the agarose gel of appropriate concentration and size
◊ 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 2: When the gel is ready, place it in the electrophoresis chamber, fill with an electrophoresis buffer and load the samples and DNA ladder and start electrophoresis. 

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

Step 3: When the electrophoresis is over, analyze the gel in the gel documentation system/uv-transilluminator.
Compare the location of DNA bands appearing in DNA samples with the known-size DNA bands of the DNA ladder.