Making and running agarose gels
TAE buffer
TAE buffer may be used for making and running agarose gels. TAE stands for Tris, Acetic Acid, EDTA. As TAE buffer can be stored at room temperature, making a 10 X concentration stock is a good time saving option opposed to continually making 1 X concentrations. To make 1L of 10 X TAE buffer (pH8) add 48.4g of Tris, 7.44g of EDTA(Na2), and 11.4mL of 100% Acetic Acid. Add deionised water to reach 1L and mix. Mixing with a magnetic stirrer is ideal. For a 1 X concentration for pouring gels and running gels a 10 X concentration may be diluted to 1 X by adding 100mL of 10 X buffer and 900mL of deionised water to a 1L bottle.
Making a 1% agarose gel
A 1% gel is good for product sizes from 100 – 1000 base pairs. Larger products can also be run on a 1% gel, though larger products will travel through the gel and seperate from one another at a slower rate.
1. For a 1% gel add 0.5g of molecular biology grade agarose powder per 50mL of 1 X TAE buffer. 50mL is usually enough for small gels. Ideal to use a microwave safe glass bottle.
2. Dissolve powder into the buffer using a microwave. Swirling the bottle a few times may be required to assist even mixing / dissolving. Make sure to wear protective eye glasses or face-shield and heat protective gloves. Make sure the mix is not bubbling before handling the bottle.
3. Cool the mix to approximately 60°C and add Ethidium Bromide. For a 50mL gel add 1.5uL of Ethidium Bromide from a 10mg/mL stock. Swirl bottle to mix the Ethidium Bromide thoroughly. (60°C ensures the Ethidium Bromide will not be damaged by excessive heat but also ensures the gel will not set before the Ethidium Bromide is mixed evenly. Ethidium Bromide binds to DNA and is used to visualise DNA in the gel under UV light. As Ethidium Bromide binds with DNA it is considered a hazardous mutagen. Make sure to wear nitrile gloves).
SYBR Safe DNA Gel Stain (from Thermo Fisher Scientific) can be used as an alternative to Ethidium Bromide.
4. Pour the gel and insert the comb. Remove any bubbles.
Running agarose gels
Run a 1% gel at 100 volts (10 volts for every 1cm of gel length) for 30 – 40 minutes for a product of 500 base pairs. Loading / tracking dye will assist in determining exactly when to complete running the gel. The best loading / tracking dyes contain multiple dyes. In 1% gel bromophenol blue runs equivalent to 300 base pairs, Orange G 50 base pairs, and Xylene cyanol 4000 base pairs.
Make sure to wear nitrile gloves when handling gels containing Ethidium Bromide.
Make sure to run a DNA ladder for verification against your expected product size. For options to consider see Bioline and Sigma-Aldrich.
