1.     Agarose Gel

 

Author: ETB Date: 3/19/01

AGAROSE GELS

For 1 gel:

Hazards:

Ethidium Bromide (EB) is a potent mutagen and moderately toxic after an acute exposure.

 

Protection:

Always follow PPE, wearing labcoat and gloves, adding EB into buffer in fume hood. Beware of using the “hot hand” when microwave Agarose gel.

 

Waste:

Tips contaminated with EB should be put into labeled container in the fume food.

Used gel should be disposed as hazardous waste.

 

Spill Clean-up

Always wear PPE during any cleanup proecedure. Absorb freestanding liquid with dry paper towels. Use a UV light to locate any remaining EB and wipe it out.

According to need, prepare 1 to 2% agarose gel. Following procedure is for 1% agarose gel:

Add 1 g powder agarose per 100 ml TAE (1X) in glass flask.

Microwave 2 mins, shake, then microwave 1 minute to be clear solution
Add 8 ml l EB solution (10mg/ml) and shake to mix well.
Pour gel and let solidify 40 min @ RT
Medium gels -> 160ml
Small gels -> 80 ml
Large gels -> 160ml x 2 (two flasks)

Samples:
Transfer 30 ml to U bottom microtiter platewell and add 5 ml dye and mix
Load the mixture onto gel
Medium teeth = 28ul, Small teeth = 24ul

For DNA weight standards:
Mix 6 ml Standard with 2 ml dye