Team:Cambridge/Protocols/Gel Electrophoresis

Gel Electrophoresis

A method to separate DNA strands of different lengths.

Theory

fig 1. Our first Gel Electrophoresis

DNA is negatively charged due to the phosphate ions (PO₄^3-) backbone. When an electric field is applied across an agarose matrix containing DNA, the nucleic acid fragments move towards the positive cathode.

This migration of DNA is dependent upon the size of the matrix pores and the length of the DNA in question. For a fixed pore size and potential difference, a particular DNA fragment migrates a distance proportional to the log₁₀ of the molecular weight of the molecule.

This allows DNA fragments to be separated by size. The sizes are calculated by comparison with a 'ladder' of standard DNA fragments of known sizes.

The distances the ladder fragments move in a given time can be plotted on a semi-log plot of molecular weight against distance to make a calibration curve which the sample fragments can be referenced against.

Practice

Gel preparation:

• For 1% agarose gel (say 200ml), add 2g of agarose powder to 200 ml of 1x TAE buffer (obtained by diluting 10x TAE stock buffer with water).

• The shorter the DNA strand lengths, the more concentrated the gel will be.
• Use 75-100ml of buffer for preparing one gel.

• Heat the mixture in the microwave until the powder has completely dissolved stirring the contents every so often.
• Transfer the solution into a disposable container.
• Gel stains should be added when the agarose becomes cool enough to touch.(For SYBR Safe gel, add 5μl to 50ml TAE buffer)

Electrophoresis setting:

• Ensure that the electrophoresis chamber is clean and dry, and tape the sides so that it is watertight. Slot in the comb.
• Pipette a small amount of the tepid gel mixture around the edges of the taped regions to seal the chamber.
• Add the remaining gel solution to the chamber, and wait for the gel to set. The cool gel can then be removed from the chamber.
• Fill the electrophoresis apparatus half-full with 1x TAE buffer solution (for good electrical contact) and place the set gel in the buffer. Ensure that there are no air bubbles (particularly in the wells created by the comb).
• Add the ladder solution to the first well, and the other mixtures to be separated to the other wells. A ?????? dye may be added to the mixtures.
• Connect the electrodes to the apparatus (the right way round!). Set DC voltage at 80V (with current at approximately 3 mA) and run for 30-60 minutes (or until the DNA has separated sufficiently).

Safety

• SYBR safe dye is dangerous to work with; everything that could have come into contact with it needs to be trated as toxic waste, including gloves (which must be worn at all times during the whole procedure).
• Liquids heated in the microwave can be superheated; a fluid that does not seem to be boiling when taken out of the microwave can boil violently when swirled. Avoid this by removing the solution from the microwave intermittently and swirling at arms length.