Recipes for common reagents used in DNA gel electrophoresis


TBE (TRIS/Borate/EDTA) - Gel and running buffer

Used for making up agarose gel and as the electrophoresis buffer
10x concentrate - Makes 1 L.
May be stored indefinitely at room temperature.

Ingredients: 

1 g Sodium hydroxide (m.m. 40.00) 
108 g TRIS base (m.m. 121.10) 
55 g Boric acid (m.m. 61.83) 
7.4 g Ethylene diamine tetraacetic acid (EDTA, disodium salt, m.m. 372.24) 


Directions:
Add the ingredients listed above to 700 mL of deionised or distilled water. 
Stir to dissolve. Make up to 1 L with deionised or distilled water. 


CAUTION

Avoid inhaling TRIS or EDTA powders. Wear a mask over your nose and mouth. Both chemicals are irritants, and can cause harm through physical contact and inhalation.

Note: Once made up, 1 volume of this TBE concentrate should be diluted with 9 volumes of distilled water before use.

IMPORTANT! DO NOT use TRIS-HCl to make up this buffer. You must use TRIS base.



Azure A
 - Stain for nucleic acids

2x concentrate - Makes 50 mL.
May be stored indefinitely at room temperature.

Ingredients:
0.08 g Azure A (m.m. 291.8) 
50 mL ethanol (40% aqueous solution)

Directions:
Add the Azure A powder to 50 mL of 40% ethanol. Stir to dissolve.

CAUTION

The concentrated DNA stain is flammable and must not be used or stored near naked flames or other sources of ignition. The stain bottle must be kept closed to prevent evaporation of the solvent. 
When diluted to its working concentration (0.04% in 20% ethanol), the stain presents no serious safety hazard, although care should be taken to avoid splashes on the skin or eyes e.g. wear protective gloves and glasses. Used stain may be diluted with water and washed down the drain.

FIRST AID (Azure A powder)
EYE CONTACT: flush eyes with copious amounts of water for at least 15 minutes. Seek medical attention. 
SKIN CONTACT: wash the skin with soap and plenty of water. If irritation occurs seek medical attention.
INGESTION: provided the person is conscious, wash out the mouth with water. Induce vomiting. Seek medical attention.

Note: Once made up, this Azure A concentrate should be diluted with an equal volume of distilled water before use.



Agarose gel - For separating small fragments of nucleic acids

0.8% agarose - Makes 100 mL.
May be stored indefinitely at room temperature.

Ingredients:
0.8 g electrophoresis grade agarose
100 mL TBE buffer
(1x - made from concentrate, above)

Directions:
Add the agarose powder to the TBE buffer. Heat in a boiling water bath or microwave oven to melt the agarose. Less than a minute at full power in a 940 watt oven is sufficient to melt 100 mL of gel. The container used to hold the molten agarose must not be sealed, but lightly covered with plastic film that has been punctured with one or two small holes. Swirl the gel halfway through the heating cycle to ensure that it is thoroughly mixed.

Once molten, the agarose solution can be kept in this state at 55–60 °C in a water bath. Ensure that the agarose solution is mixed thoroughly before casting gels.

CAUTION

Hot, molten agarose can scald and so it must be handled with care. It is advisable to let the molten agarose cool until it is comfortable to handle before pouring the gel.



Bromophenol blue loading dye - Used for loading DNA fragments into the electrophoresis gel

Makes 100 mL.
May be stored indefinitely at room temperature.

Ingredients:
0.25 g Bromophenol blue (m.m. 669.96)
50 g Sucrose (m.m. 342.30)
1 mL 1 M TRIS (pH 8, m.m. 121.10)

Directions:
Add the ingredients listed above to 60 mL of deionised or distilled water. 
Stir to dissolve. Make up to 100 mL with deionised or distilled water.