Oneill Lab:Gels

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

Contents

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
  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]

Lab Homepage

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