Home | Jobs | News & Articles | Job Advice | Search | Protocols | Fun | @RealLabRat
Lab Rat with DNA

Post your resume
Candidates - post your resume

Search job listings
Candidates - search biotech jobs

Post job openings
Employers - post job openings
HOME > Protocols > Molecular Biology > Oligonucleotide > Oligonucleotide Purification Protocol

Oligonucleotide Purification Protocol

  1. Prepare 1M TEAA:
    ----Add 5.7ml glacial HOAc to 50ml H2O and mix well
    ----Add 13.9ml triethylamine and mix well -add H2O to final volume of 100ml and mix well
    ----Adjust pH to 7.0 with triethylamine or HOAc and store in tightly capped light protective container at +4oC
  2. After synthesis of oligonucleotide, deprotect in 30% NH4OH at room temperature for 24 hours
  3. Pass 3 X 5ml of 100% acetonitrile over column (we used MilliGen/Biosearch Oligo-Pak columns)
  4. Flush column with 3 X 5ml 1M TEAA
  5. Dilute oligonucleotide 1:1 with deionized H2O and collect flow through, repeat twice
  6. Wash column with 3 X 5ml 3% NH4OH
  7. If oligonucleotide is 75mer or less, wash column with 3 X 5ml deionized H2O; if greater than 75mer wash column with 4 X 5ml deionized H2O
  8. Wash column with 5ml 2% TFA
  9. Wash column with 5ml deionized H2O
  10. Elute oligonucleotide from column with 3 X 1ml 20% acetonitrile
  11. Read a sample of eluted oligonucleotide at A260/A280 to estimate total recovered material and freeze remaining sample at -70oC
  12. Lyophilize frozen oligonucleotide eluent overnight in refrigerated speedvac and resuspend pellet in appropriate volume of deionized H2O
  13. Read sample of final oligonucleotide at A260/A280

theLabRat.com 2005. All Rights Reserved.