RRC-PRL: High Performance Ion-Exchange Purification of Oligonucleotides

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Contents

Summary

High Performance Ion-Exchange Purification of Oligonucleotides Trityl-off oligonucleotides are purified with Vydac 301VHP anion exchange columns. These columns consist of tertiary amine functional groups attached to modified polystyrene-divinylbenzene beads. 301VHP columns are stable in both base and acid (pH 1-13). The separation is based on the number of the phosphate groups.

Oligonucleotide reaction products are eluted with a salt gradient near neutral pH. The product of interest generally elutes as the last major peak. Earlier eluting peaks are minus-mers and other reaction by-products. High performance ion exchange chromatography can purify detritylated oligonucleotides up to 60-mer.

General set up and conditions

For 10-mer. Column: Vydac 301VHP575 (DEAE weak anion exchange 5m . 7.5mmlD x50mmL, 900 angstroms pore diameter). Eluent: 10 mM Tris-HCL, pH 8.0, gradient from 0 to 0.5M NaCL in 35min.

For 25-mer. Column: Vydac 301VHP575. Conditions: 1.0ml/min. 10 mM Tris-HCL, pH 8.0, gradient from 0 to 0.2M NaCL in 5minutes, then 0.2-0.3M NaCL in 40 min.

Column- P/N 301VHP575 VYDAC

Gradient
time flow rate
(ml/min)
%A %B
2.0 1.0 97.0 3.0
2.0 1.0 97.0 3.0
60.0 1.0 0 100.0
5.0 1.0 0 100.0
10.0 1.0 97.0 3.0
0 0 0 0


Buffer A - 10 mM Tris-HCL, pH 8.0; Buffer B - 10 mM Tris-HCL, 1M NaCL, pH 8.0

We can inject up to 5mL of a sample. The sample should be dissolved in buffer A or equivalent solvens.

After finish, wash column with water and store the column in water or 0.02% (w/v) sodium azide in water.

Column regeneration

VHP575 (2.2 ml column volume) VHP columns are resistant to attack by most common organic solvents and by most acids and bases and can be cleaned by treatment with any of several solutions.

  • For strongly bound hydrophilic proteins: Inject 0.5-1.0mL of 2M NaCl while rinsing with a strong ionic-strength buffer such as used at the end of a gradient.

For strongly bound hydrophobic proteins: Rinse the column with 10-20 column volumes of isopropanol after first removing salt from the column with 10 column volumes of water.

For bound lipids: Rinse the column with chloroform or dichloromethane after fist removing salts with 10 column volumes of water followed by 10 column volumes of methanol.

For other general cleaning: We recommend rinsing the column with one or more of the following: 10 column volumes of 0.5N NaOH; 10 column volumes of 0.5N HCl; or 10 column volumes of 50% acetic acid.

Peptide purification

TFA Cleavage Procedure

The following procedures are designed for cleavage of 0.1 to 1.5g of peptide-resin.

Cleavage mixtures:

 A
    0.5ml D.I. water
    9.5ml TFA
 B
    0.75g crystalline phenol
    0.25ml EDT
    0.5ml thioanisole
    0.5ml D.I. water
    10ml TFA
 C
    0.25ml EDT
    0.25ml water
    9.5ml TFA
  1. Prepare the appropriate cleavage mixture- either A, B or C- as indicated by the Fmoc Cleavage Flow Chart.
  2. Place the dried peptide- resin in a 20ml glass bottle or 50ml plastic tube that contains a small stir bar.
  3. Add the cleavage mixture to the peptide-resin to give a total reaction volume of 10ml per 0.1-1.5g of peptide- resin. Stopper the tube and stir the reaction mixture at room temperature for 2hrs.
  4. After the reaction time has elapsed, filter the reaction mixture through a medium- porosity plastic filter (0.1 mm Pore Size Whatman PTFE Filter Tube) into another 50ml tube to separate the peptide solution from the resin support.
  5. Add cold ethyl ether to the reaction mixture to precipitate the peptide.
  6. Centrifuge for 10 minutes.
  7. Discard the supernatant and resuspend the precipitate in 50ml ethyl ether with vortex mixing. Centrifuge again. This process can be repeated for several times.
  8. Dry the peptide in fumehood.
  9. Dissolve the peptide for lyophilization or HPLC purification.

HPLC peptide purification

Synthetic peptides are purified with Vydac cat # 218TP1010 Protein & Peptides C18 column (5m , 10x250mm).

Conditions:

Gradient
time flow rate
(ml/min)
%A %B
2.0 5.0 97.0 3.0
2.0 5.0 97.0 3.0
60.0 5.0 0 100.0
5.0 5.0 0 100.0
10.0 5.0 97.0 3.0
0 0 0 0


Buffer A- 99.9% water, 0.1% TFA Buffer B- 99.9% acetonitrile, 0.1% TFA

We can inject up to 5mL of a sample. The sample should be dissolved in buffer A or equivalent solvens.

After finish , wash column with 50% acetonitrile.

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