This protocol outlines how to perform TBE-Urea PAGE purification by electrophoresis with the crush-and-soak method.
| Item | Item article number | Distributor | Vendor |
|---|---|---|---|
| TBE-Urea PAGE precast gels 10% or 15% | EC6885BOX | ThermoFisher | Invitrogen |
| 10x TBE buffer | |||
| Hi-D formamide | 4311320 | Applied Biosystems | ThermoScientific |
| 10x 1M sodium acetate buffer | |||
| Pierce Spin Cups | 69700 | ThermoScientific | ThermoScientific |
| Amicon Ultra 0.5 3K | UFC500324 | Merck Millipore | SigmaAldrich |
Set up the TBE-Urea gel (10% or 15%) to run in 1x TBE buffer. Use a syringe to clean each well.
Run the gel for 1h at 300 Volt before loading the gel. This helps even the temperature in the gel and help denaturing.
Load a maximum of ~1 µg per well (for 9mer ssDNA mw ~2718.6 g/mole).
The molecular weight, mw, of an oligo can roughly be approximated by its length, nt: mw = 308.95×nt - 62
| Reagent | Amount | Final concentration |
|---|---|---|
| Sample (16'311.6 ng) | 30 µl | 163.116 ng/µl |
| Formamide | 70 µl | 70% |
| Total: | 100 µl |
Load 2-10 µl per well and run the gel until bands are clearly distinguishable to be cut out.
Use a razor blade to cut out the upper 1/2 to 2/3 of the band of interest.
Transfer the gel slice to a microcentrifuge tube. Use a sterile glass rod to crush the slice against the wall of the tube.
Add 500 µl of 0.1 M sodium acetate (pH 6.0).
Incubate at 90°C for 5 min.
Push the tube down onto dry ice (-70°C), or into a -80°C freezer. Incubate for 5 min.
Thaw and transfer to a Pierce Spin Cups paper or nylon filter.
Spin for 30 min at 14,000 x g.
Transfer the flowthrough Amicon Ultra-0.5 Centrifugal Filter Unit (3 kDa cut-off for short oligos).
Spin for 30 min at 14,000 x g.