Team:Cambridge/Protocols/GelElectrophoresis

Gel Electrophoresis

A method to separate DNA strands of different lengths.

Theory

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:

1. 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).
   
   • Note: The shorter the DNA strand lengths, the more concentrated the gel will be.
   • Use 75-100ml of buffer for preparing one gel.
2. Heat the mixture in the microwave until the powder has completely dissolved stirring the contents every so often.
3. Transfer the solution into a disposable container.
4. 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:

1. Ensure electrophoresis chamber is clean and dry, tape the sides (with Autoclave tape, NOT standard masking tape) to make watertight. Slot in the desired comb.
2. Pipette a small amount of the tepid gel mixture around the edges of the taped regions to seal the chamber.
3. Add remaining gel solution to the chamber, and wait to set. The comb can then be removed from the chamber.
4. 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).
5. Add the ladder solution to the first well, and the DNA samples to subsequent wells. A loading dye may be added to the mixtures to aid visualisation when loading into wells.
6. 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).

Tips for a Successful Gel

• Add buffer, not water, when making the gel
• Seal the gel mould using autoclave tape (not masking tape) and with hot agarose
• After boiling buffer and agarose, let it cool before pouring into mould to prevent leakage
• Use running buffer to lubricate removal of mould else risk breaking the wells
• High salt is bad so dilute sample after enzymatic reactions
• Use full volume of well
• Check DNA is running towards the positive/cathode/red pole
• Check that your voltage and current are appropriate; running gel too fast will distort the bands
• Use fresh buffer for each gel, as a pH gradient will build up during each run

Safety

• SYBR safe dye is dangerous to work with; everything that could have come into contact with it needs to be treated as hazardous chemical 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.