1.Add agarose to 1X TBE (or TAE) buffer. For gel size 20 x 24 cm, use 300-400 ml buffer and 0.7 to 1.0% agarose.
2. Melt agarose in 500 ml flask in microwave oven, mixing several times during heating. Let cool to 55 C (until flask can be held).
3. Tape the ends of gel tray and place on a level bench.
4. Add ethidium bromide: 2.5 ul of 10 mg/ml stock per 100 ml. (gel cam also be stained in ethidium bromide bath after electrophoresis (see point 9).
NOTE: Ethidium bromide is mutagenic - wear gloves when handling, and use extra caution. Change gloves when contaminated and dispose in separate waste for ethidium bromide.
5. Pour agarose into tray and insert combs. Remove bubbles with a pipette tip. Allow to solidify.
6. Remove tape and place tray in gel boxes. Pour enough 1X TBE (or TAE) buffer into the gel box to cover the gel by at least 0.5 cm. Remove combs when ready to load samples.
7. Load 1 ug Lambda digested with Hind III (5 ul of 200 ng/ul stock) as molecular weight marker, then load samples.
8. Run at 15-25 V for 12-24 hours.
9. If no ethidium bromide was added to the agarose: stain gel in 1 ug/ml ethidium bromide (100 ul of 10 mg/ml ethidium bromide in 1000 ml dd H2O) for 20 min. 10. Rinse gel for 20 min in 1000 ml ddH2O.
11. Slide gel onto UV transilluminator and take photo.
Place small piece of paper with writing or transparent ruler on the gel to help focus.
High resolution agarose
MetaPhor agarose is a high resolution agarose that challenges polyacrylamide. MetaPhor agarose is an intermediate melting temperature (75° C) agarose with twice the resolution capabilities of the finest-sieving agarose products. Using submarine gel electrophoresis, you can resolve PCR products and small DNA fragments that differ in size by 2%.
Gelling temperature (3%) = 35° C
Melting temperature (3%) = 75° C
Gel strength (3%) = 300 g/cm²
• High resolution separation of 20-800 bp DNA fragments
• Recovery of fragments under 800 bp
• Fine analysis of PCR? products
• AMPFLP, STR and tri- and tetranucleotide repeat analysis
Suggested Agarose Concentrations
Dye Mobility Table
Migration of double-stranded DNA in relation to Bromophenol Blue (BPB) and Xylene Cyanol (XC) in MetaPhor agarose gels.
Always wear eye protection when dissolving agarose and guard yourself and others against scalding solutions.
Microwave Instructions for Agarose Preparation
1. Choose a beaker that is 2-4 times the volume of the solution.
2. Add chilled 1X or 0.5X electrophoresis buffer and a stir bar to the beaker.
3. Slowly sprinkle in the agarose powder while the solution is rapidly stirred.
4. Remove the stir bar if not Teflon® coated.
5. Soak the agarose in the buffer for 15 minutes before heating. This reduces the tendency of the agarose solution to foam during heating.
6. Weigh the beaker and solution before heating.
7. Cover the beaker with plastic wrap.
8. Pierce a small hole in the plastic wrap for ventilation.
For agarose concentrations > 4%, the following additional steps will further help prevent the agarose solution from foaming during melting/dissolution:
a. Heat the beaker in the microwave oven on Medium power for 1 minute.
b. Remove the solution from the microwave.
c. Allow the solution to sit on the bench for 15 minutes.
9. Heat the beaker in the microwave oven on Medium power for 2 minutes.
10. Remove the beaker from the microwave oven. Caution: Any microwaved
solution may become superheated and foam over when agitated.
11. GENTLY swirl the beaker to resuspend any settled powder and gel pieces.
12. Reheat the beaker on HIGH power until the solution comes to a boil.
13. Hold at boiling point for 1 minute or until all of the particles are dissolved.
14. Remove the beaker from the microwave oven.
15. GENTLY swirl the beaker to thoroughly mix the agarose solution.
16. After dissolution, add sufficient hot distilled water to obtain the initial weight.
17. Mix thoroughly.
18. Cool the solution to 50-60°C prior to casting. Once the gel is cast, allow the molten agarose to cool and gel at room temperature. The gel must then be placed at 4° C for 20 minutes to obtain optimal resolution and gel handling characteristics.
Hot Plate Instructions for Agarose Preparation
1 . Choose a beaker that is 2-4 times the volume of the solution.
2. Add chilled electrophoresis buffer and a stir bar to the beaker.
3. Slowly sprinkle the agarose powder while the solution is rapidly stirred.
4. Weigh the beaker and solution before heating.
5. Cover the beaker with plastic wrap.
6. Pierce a small hole in the plastic wrap for ventilation.
7. Bring the solution to a boil while stirring.
8. Maintain gentle boiling until all the agarose is dissolved (approximately 10 minutes).
9. Add sufficient hot distilled water to obtain the initial weight.
10. Mix thoroughly.
11 . Cool the solution to 50-60°C prior to casting. Once the gel is cast, allow the molten agarose to cool and gel at room temperature. The gel must then be placed at 4° C for 20 minutes to obtain optimal resolution and gel handling characteristics.
These products may be used as in vitro medical devices inaccordance with the U.S. Food, Drug, and Cosmetics Act.