Oneill Lab:Gels

Michael J. O'Neill Lab University of Connecticut Department of Molecular and Cell Biology

Agarose gels

• Electrophoresis grade agarose gels are made with TBE
• NuSieve gels for DNA purification are made with TAE

Formaldehyde gel

For making a 1% 100 mL

1. Combine
   • 5mL 20x Running Buffer
   • 77mL deionized water
   • 1g agarose
2. Boil in microwave until agarose dissolves
3. Add 18mL 37% formaldehyde
   • Swirl flask while pouring
   • Add formaldehyde in fume hood
4. Pour gel in fume hood

Polyacrylamide gels

For resolving nucleic acids

	10%	7.5%	5%
Acrylamide	8.3 ml	6.25 ml	4 ml
1x TBE	16.4 ml	18.5 ml	20.8
10% APS	250 μL	250 μL	250 μL
TEMED	25 μL	25 μL	25 μL

One-dimensional denaturing gel for resolving peptides

5 ml stacking gel (5%) 20 ml resolving gel	5%	8%	9%	10%	11%	12%
Range of separation (kD)		40-100	30-90	20-80	16-70	12-60
H2O (ml)	3.4	9.3	8.6	7.9	7.2	6.5
29:1 Acrylamide:bis-acrylamide Protogel (mL)	0.83	5.3	6.0	6.7	7.4	8.0
1.5M Tris pH 8.8	0.63	5.0 mL	5.0 mL	5.0 mL	5.0 mL	5.0 mL
20% SDS (μL)	25	100	100	100	100	100
10% Ammonium Persulfate (μL)	50	200	200	200	200	200
TEMED (μL)	15	15	15	15	15	15

Add APS and TEMED last. Gel will begin polymerizing immediately.

1. Pour resolving gel first. Be sure to leave space for stacking gel. Will need to allow for length of your comb + 1 cm.
2. Once resolving gel is poured, cover in a layer of iso-butyl alcohol. Leave gel vertical while it polymerizes. This usually takes 45-60 minutes
3. After the gel polymerizes, pour off the alcohol, and rinse the exposed gel with deionized water. Use a paper towel to wick away as much water as possible.
4. Prepare and pour stacking gel to top of plate. Add comb and allow gel to polymerize for 1 hour.

Links and Contacts

Seth Kasowitz [1]

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