Kenneth A. Johnson, Ph.D Lab | Methods

 

PAGE Purification of Oligos for Kinetic Assays

Written by: Matthew Kellinger
Version 2010-06-07

Note: For best results (and to not have to repeat this tedious procedure multiple times per year) order a 1µmol scale oligo from IDT with standard desalting.

  • 1) Spin IDT tube containing lyophilized oligo to ensure the contents reach the bottom of the tube.
  • 2) Add 2mL of PAGE purification buffer (Deionized Formamide + Bromophenol Blue ONLY! (No Xylene Cyanol which runs at the same speed as most oligos and intereferes with UV backshadowing)
  • 3) Heat Oligo to 95°C to denature.

Casting and Running a Denaturing PAGE Gel:

  • 1) Select the correct percentage of Acrylamide based on the size of oligo you are purifying (Look in the red book pg. 2.12.2)
  • 2) Mix 300mL of Casting Solution
    • a. XX% w/v Acrylamide (19:1 Acrylamide : Bis)
    • b. 7M Urea (MW=60.06) 126.1g for 300mL
    • c. 1× TBE (from 10x Stock)
    • d. Bring Volume to 275mL by eye in beaker
    • e. Mix solution on stir plate in ice bucket filled with warm water
  • 3) Bring dissolved gel solution up to 300mL in graduated cylinder and filter thru Whatman 4 filter paper
  • 4) Assemble PAGE purification apparatus with casting reservoir
  • 5) Polymerize 75mL of the gel solution with 375µL of Fresh 10% APS and 75µL TEMED. Pour into the bottom casting tray and lean the apparatus upright.
  • 6) After polymerization has occurred, polymerize the remaining gel solution by adding 1125µL 10% APS and 225µL TEMED in as small of a beaker as possible.
  • 7) Pour the Acylamide solution from the top of the apparatus. Use gravity to assist by keeping a slight angle from top to bottom and side to side. Allow the solution to flow down the side of the apparatus to ensure no bubbles.
  • 8) Once poured, lay the apparatus flat on the benchtop to prevent leaking and insert the comb. Allow 90 minutes to polymerize and cure.
  • 9) Remove the bottom casting reservoir and knock off as much of excess acrylamide as possible. Dispose of properly.
  • 10) Place the gel into the running position. Add 1x TBE to the top and bottom of the tank.
  • 11) Heat gel using constant wattage = 60W until the gel reaches 50°C.
  • 12) Remove the comb and blow out the wells that will have pooled Urea.
  • 13) Add 200µL of Oligo to each of the 5 wells and run until the dye front reaches the bottom. Keep the remaining 1000µL for future purification.

Recovering the Purified Oligo:

  • 1) After disassembling the gel equipment, transfer the gel to a piece of plastic wrap.
  • 2) UV backshadow the gel using the short wavelength hand-held UV lamp (CAREFUL: It doesn't take long to get a sunburn on your hands)
  • 3) Cut out bands being careful to avoid any n-1 product of synthesis (sacrifice some full length oligo if necessary).
  • 4) Excise bands and place gel slices into a 15mL Falcon Tube.
  • 5) Crush acrylamide with a glass rod to increase surface area, then add 10mL of 1x TE. The oligo will reach equilibrium between the gel and the buffer. Freeze for 1 hour at -80.
  • 6) Heat at 65°C for 1 hour.
  • 7) Filter the solution thru syringe filters to remove the acrylamide (push hard or use multiple filter cassettes).
  • 8) Add and equal volume of 1-Butanol to the falcon tube under the hood. The 1- Butanol will dehydrate the solution, which concentrates the oligo.
  • 9) Repeat step 8 until the aqueous volume is ~500µL. If too much 1-butanol is added and the aqueous layer (bottom) disappears, add more TE and start step 8 over.
  • 10) Recover concentrated oligo to a 2mL Tube.
  • 11) Ethanol Precipitate by adding 1/10th volume 8M LiCl and 2.5volumes 100% Ethanol. Store in -20°C for 20 minutes.
  • 12) Centrifuge the Tube for 20 minutes at 13krpm, 4°C.
  • 13) Discard supernatant and wash the pellet with 1mL of 70% Ethanol to remove excess salt.
  • 14) Place at 37°C until the pellet is DRY (Don't cheat of or your oligo will have a large absorbance at 230nm).
  • 15) Resuspend the dry oligo in 300µL of 1x 66.2 buffer. Record OD260 to determine yield and dilute the final oligo to a 100uM working stock.