TBE-UREA PAGE Gel
6% PAGE Stock Solution (50 mL)
|
Reagents |
Volume |
| 40% Acrylamide : bis-acrylamide (19:1) | 7.5 mL |
| 10X TBE | 5 mL |
| Urea | 21 g (Final Conc. = 7M) |
Top up to 50 mL with MPW.
Individual Gel (10 mL)
| Reagents | Volume |
| 6% PAGE stock | 10 mL |
| 10% APS | 100 µL |
| TEMED | 10 µL |
- %T = %A + %B = 40%
- %C = %BA/%A + %B = 1 / (1+19) = 5%
[Sample Preparation and the Electrophoresis Condition]
Running Buffer = 1x TBE. Takes 450 mL to fill the mini tank.
Loading Buffer: TO make 10 mL
| Reagents | Volume |
| Formamide | 10 mL |
| Bromophenol Blue | 2 mg |
- Mix the loading dye and the ssDNA (or RNA) sample in 1:1 ratio.
- Boil the dsDNA ladder and the ssDNA sample mixtures at 95˚C for ~5 min, and then put on ice before loading on the denaturing gel.
- Load about 10 microliters of the mixture to each well. (Loading Dye: load about 3 microliters.)
- Run the gel at the room temperature at 200V for 30min. In this way, as the electrophoresis takes place, heat will be generated. Heat is important for the denaturing urea gel. Heat will denature the DNA, so that the samples will run based on their molecular weights.
[Staining]
- Make desired volume of the staining soluiton: 1X TBE + 1/10,000 Syber Gold stain. For a small gel, 15-20 mL would be a good starting point.
- Stain the gel for ~30 min or longer if needed, and take the gel image.