🧪 PAGE purification

This protocol outlines how to perform TBE-Urea PAGE purification by electrophoresis with the crush-and-soak method.

🚛 Reagents needed 📦 :
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, ntmw = 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.

⛏️💥 💦 'Crush-and-Soak' recovering 🎣 of oligonucleotides from Polyacrylamide Gel 🍮

 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.