OLIGO PURIFICATION on acrylamide gels

Friday, November 21, 2003

Description
OLIGO PURIFICATION on acrylamide gels

Procedure
GEL PURIFICATION OF OLIGONUCLEOTIDES

1. Quantitate crude oligo solution via UV spectrophotometry. Assume 1.0 A260 unit is equal to 33 ug/ml. Dry down 100ug of oligo on speedi-vac overnight.

2. Resuspend dried-down oligo in 40ul TE. Add 20ul formamide dye.

3. Prepare a 12% polyacrylamide gel (20:1 acrylamide:bis-acrylamide).

6.3ml 20x TBE

32ml 7M urea

38ml 40% acrylamide/7M urea

50ml dH20

375 ul ammonium persulphate

75ul TEMED

4. Cast gel in 25 x 25 cm plates with 0.75mm spacers. Rainex the plate with ears. Use combs with >8mm teeth. It is important to flush the wells very thoroughly prior to pre running and loading oligos onto the gel. A build-up of urea in the well will prevent the oligo running into the gel. Pre-run gel at 400v for 15 minutes. Load samples (100ug per three of four wells) and run gel at 400v until bromophenol blue is two-thirds down the gel.

5. Lay gel ears side up and prize off top plate. Cover gel with glad wrap and turn plate over. Gently lift off remaining glass plate, leaving gel on the glad wrap.

6. Place gel onto a TLC plate and visualise bands by UV shadowing. Bands should appear purple on the green background of the TLC plate. Excise band with a razor blade and place into a 10ml tube with 1ml TE. Allow to elute overnight with agitation (Optional: break up gel slice by passing through a stop cock from one syringe to another before eluting over night).

7. Filter the eluate through a pre-wetted acrodisk. Collect eluate and add 5ml butanol. Vortex for 30 seconds and spin at 3500 rpm for 5 minutes. Remove organic (top) phase and place aqueous phase (should be ca. 100-200ul) into an eppendorf. Adjust volume to 500ul with TE. Phenol/chloroform and chloroform extract. EtOH precipitate and spin 13000 rpm for one hour. Wash pellet in 100ul 70% EtOH.

8. Resuspend pellet in 25ul TE and quantitate.

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OLIGO PURIFICATION on acrylamide gels