General DNA Agarose Gel Electrophoresis protocol

Background

This protocol is suitable for electrophoresis separation of DNA fragments from PCR reactions.

Solutions

• 10X TBE, for 1 l:

Component	Weight	Concentration
Tris base	121.1 g	1M
Boric acid	61.8 g	1 M
EDTA (disodium salt)	7.4 g	0.02 M

• 6X DNA loading dye, for 10 ml:

Component	Amount	Concentration
Glycerol	3 ml	30 %
Bromophenol blue	25 mg	0.25%

Make to 10 ml final volume.

Materials

• Horizontal electrophoresis unit with power supply
• Conical flask for gel preparations
• Heat (microwave or hotplate)
• Gel combs

Methods

Gel preparation

• Dilute 10X TBE buffer to make a 1X solution (100 ml in 900 ml H₂O).
• Add 100 ml of 1X TBE to a conical beaker. Add agarose to create a suitable percentage gel (2 g for 2% gel).
• If using Sybr Safe gel stain, add the stain 1:10000 to the mixture (10 ul in 100 ml)
• Heat the mixture until the solution gets clear (e.g. 1 min in microwave or on a hotplate).
• Let the mixture cool to about 60 degrees (it should feel warm but not hot).
• Pour the mixture into a gel casting tray and place the comb at its designated place.
• The gel will polymerize in 1 h, remove the comb after polymerization.

Running the gel

• Place the gel in the electrophoresis unit.
• Pour 1X TBE into the electrophoresis tank to cover the well.
• Mix samples with loading dye (1 ul of 6X dye per every 5 ul of sample).
• Add DNA ladder and samples to the wells
• Set voltage to 150 V, run four about 1 h (until dye is about 80% through), check that samples moves towards the positive elctrode!

Visualisation

• Visuialize the gel i G:Box using the UV light emitter and Sybr green settings.