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
1.0M Tris pH 6.8	0.63 mL
1.5M Tris pH 8.8		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.