Denaturing PAGE

(Adapted from Bryan Harada’s protocol, 02/25/12).

for Extraction of single stranded DNA probes

Materials:

• 2x Loading Buffer (96% formamide/20mM EDTA, prepare using freshly deionized formamide)
• Acrylamide/Bis 19:1 40% (w/v) Solution  
• TEMED
• 10% APS (w/v). make fresh from powder and store frozen aliquots (<3 months)
• 1-Butanol

Procedure:

1. Assemble gel plates and seal with the gel sealing tape.
   1. Prepare 100 mL of acrylamide solution. For a 12% gel:
   • Urea 48 g
   • 40% Acrylomide 30 mL
   • 10x TBE buffer 10 mL
   • ddH2O 20 mL
   1. When urea is dissolved, add 150µL of TEMED and 300mL of 10% APS. Mix thoroughly, then pour between gel plates.
   2. Insert comb, and let gel polymerize for > 45 min.
   3. Remove sealing tape and insert gel into gel running apparatus. Fill with 0.5x TBE.
   4. Pre-run gel at 50mA for > 1hr.
   5. Dissolve the precipitated oligos in 30µL TE and add an equal volume of 2x loading buffer.
   6. Heat samples for 5 min at 95°C.
   7. Load samples onto gel and run gel at 50mA.
   8. Cut out band, crush using pestle, add to a 15mL Falcon tube add 4mL of nuclease free H2O (or TE) then incubate overnight at 37°C with shaking. [PAUSE POINT]
   9. Spin down PAGE extraction, save supernatant, and extract gel fragments with an additional 2 mL of nuclease free H2O (or TE) for >2 hrs.
   10. Spin down PAGE extraction and combine with previous supernatant.
   11. Perform butanol extractions
   • add 3 volumes of 1-BuOH to the sample,
   • vortex to mix,
   • spin briefly to separate layers (1000 RPM 30s),
   • discard organic [top] layer (butanol-waste).
   • Repeat until the volume is < 300 µL.
   1. If residual polyacrylamide fragments are present, centrifuge aqueous layer to remove gel fragments.
   2. Ethanol precipitate the DNA:
   • add 1/10 volumes of 3M NaOAc, mix,
   • add 2.5 volumes (including the NaOAc) ice-cold EtOH.
   • Incubate at -20°C for > 30 min, then spin for 30 min at max speed in cold room.
   • Pour off supernatant, add 0.5 mL of ice-cold 80% EtOH to pellet, spin for 5 min at room temp, and pour off supernatant.
   • Dry pellet by air drying or using the speed vac. Do not overdry single-stranded DNA (may degrade).