Glen Report 21.12: Procedure for Synthesis of CleanAmp™ Primers

ReagentsVendor
CleanAmp™ DNA phosphoramidites TriLink BioTech
Glen Research
UltraMild DNA phosphoramidites Glen Research
Glen UnySupport Glen Research
Acetonitrile, ≤ 20 ppm water Glen Research
Dichloromethane Fisher Scientific
Activated Molecular Sieves TriLink BioTech
UltraMild Cap A Glen Research
Cap B with N-Methyl Imidazole Glen Research
HPLC Grade Water Fisher Scientific
0.05M Potassium Carbonate in Methanol Glen Research
1M TEAA, pH 7.2 TriLink BioTech
2M TEAA, pH 7 - dilute to concentration Glen Research
2% TFA in water TriLink BioTech
Glen Research
DMSO anhydrous Fisher Scientific
Sep-Pak® Cartridges (Part # 20515) Waters

Procedure:

A. Synthesis of CleanAmp™ oligonucleotides on solid support at 1 µmole scale:

  1. Prepare solutions of each fast UltraMild phosphoramidite in anhydrous acetonitrile (ACN) using manufacturer’s instructions. We recommend using a concentration of 0.067M.
  2. Prepare 0.067 M solutions of each CleanAmp™ phosphoramidite (except for CleanAmp™-dG) in dry acetonitrile in a standard DNA synthesizer bottle. Prepare 0.067 M solution of CleanAmp™-dG phosphoramidite monomer in mixture of dry dichloromethane (DCM):ACN (1:1) in a standard DNA synthesizer bottle. To properly dissolve a 100 umole bottle of a CleanAmp™monomer to obtain a 0.067 M solution, use 1.50 mL of the appropriate solvent.
  3. Add activated molecular sieves (20 beads/mL) to each amidite bottle, flush with dry argon gas, recap the bottle, seal using parafilm and keep it overnight at room temperature before use.
  4. Purchase, or manually fill, a 1 µmole synthesis column with Glen UnySupport.
  5. Load the DNA synthesizer with the reagents listed in the Reagents table in the appropriate port according to manufacturer’s instructions.
    1. Load the amidites. Use a spare port for the CleanAmp™ monomer(s).
    2. Load the ancillary reagents. It is critical that the appropriate capping reagents are used for fast deprotecting monomers. “Cap A” must be the phenoxyacetyl anhydride version of the reagent. DO NOT USE ACETIC ANHYDRIDE.
  6. Follow an automated synthesis protocol for fast deprotecting phosphoramidite monomers as recommended by the instrument manufacturer with the following exception: increase the coupling time for CleanAmp™ phosphoramidites to ten minutes.
  7. At the last coupling cycle, leave the DMT group “On”.
  8. When synthesis is completed, dry the column using argon flow.
  9. It is recommended to proceed with the cleavage/deprotection of CleanAmp™ oligonucleotide immediately after completion of the synthesis, otherwise keep the dry column at –20°C.

B. Cleavage/Deprotection of CleanAmp™ oligonucleotides:

  1. Transfer support (with oligonucleotide) from the column to a screw cap 8 mL glass vial.
  2. Add 6 mL of 0.05M potassium carbonate (K2CO3) in MeOH.
  3. Place the vial on a rotary mixer for 20 hrs at room temperature.
  4. Let support settle and transfer supernatant solution into a 50 mL conical tube.
  5. Wash support with 2 mL of 1 M TEAA and add to the deprotection solution.
  6. Measure a total crude yield using UV spectrophotometer.
  7. Process the solution immediately using Sep-Pak isolation/purification, otherwise keep the solution at –80°C to –20°C. C.

Sep-Pak® isolation and purification procedure:

  1. Fit a 60 mL syringe to the appropriate Sep-Pak cartridge part # 20515.
  2. Equilibrate cartridge with 10 mL of acetonitrile, followed by 10 mL of 50% acetonitrile in 0.1M TEAA and finally by 15 mL of 0.025M TEAA.
  3. Prepare sample for loading:
    1. Add 2 mL of 1 M TEAA to the sample and pipette the solution into 5 mL syringe fitted with a luer lock and a 0.44 micron filter disc. Filter the solution into a 50 mL conical tube.
    2. Rinse the deprotection vessel with another 2 mL of water, passing that through the filter and into the tube with the sample also. c. Dilute the solution to 50 mL with 1 M TEAA.
  4. Load the solution onto cartridge with a flow rate of 1-2 mL/min using an appropriately sized syringe. Collect and read the absorbance of the flow-through to ensure the oligo is bound to cartridge.
  5. Rinse the cartridge with 20 mL of water.
  6. Pass 10 mL of 2% TFA through the cartridge over 3 minutes. Observe the appearance of an orange colored band.
  7. Immediately rinse the Sep-Pak with 20 mL HPLC water.
  8. Pass 10 mL of 1 M TEAA through the cartridge over 5 minutes to neutralize acid.
  9. Wash Sep-Pak with a purification buffer: a. For CleanAmp™ Turbo Oligos, apply 15 mL of 15% ACN, 0.1M TEAA to cartridge. Collect rinse. b. For CleanAmp™ Precision Oligos, apply 15 mL of 25% ACN, 0.1M TEAA to cartridge. Collect rinse.
  10. Rinse cartridge with 20 mL of water. Collect rinse.
  11. Elute samples using DMSO.
    1. Attach a 3mL syringe to the cartridge.
    2. Add 200 µL DMSO to syringe and elute fraction 1 into a microtube.
    3. Remove syringe from cartridge, pull out plunger, reattach the syringe to the cartridge, and push air into same graduated tube to completely remove all the DMSO.
    4. Add 200 µL DMSO to syringe and elute into a fresh microtube (fraction 2) using plunger. Add another 200 µL to DMSO to syringe and elute into the same graduated tube for a total of 400 µL DMSO in fraction 2.
    5. Add 200 µL DMSO to syringe and elute into a fresh microtube (fraction 3).
  12. Cap, vortex and spin down fractions. Read fraction 2 only. If yield is less than 10 OD260 units, read the absorbance of fraction 3. Add just enough of the material in fraction 3 to the material in fraction 2 to achieve 10 OD260 units.
  13. Determine molar concentration of purified oligonucleotide in DMSO solution using the absorbance reading and the calculated extinction coefficient. If necessary, dilute with DMSO to obtain a 0.2 mM solution.
  14. Analyze purified CleanAmp™ oligonucleotides by RP-HPLC to ensure that no more than 1-2% of unmodified primer is in the purified sample.
  15. Store the oligonucleotide at 4°C or less. Although it is very stable at room temperature in the DMSO solution, long term storage is improved at lower temperatures.

HPLC Analysis of CleanAmp™ Primers

Figure 1
Figure 2: structures Of CleanAmp™ Turbo and Precision Primers
  1. Analyze a 0.2 OD260 unit sample of the isolated material by reverse phase HPLC to determine the level of contamination by oligonucleotides not protected by the CleanAmp™ moiety.
  2. Method:
    1. Column: Waters µBondapak 8 mm RP cartridge (WAT 027324)
    2. Buffer A: 100 mM TEAA; Buffer B: ACN
    3. Gradient: 0-100% B over 40 minutes
    4. Flow Rate: 1 mL/minute
    5. Observe at 260 nm
  3. Determine the mobility of the unprotected oligonucleotide by heating another 0.2 OD260 unit sample for 40 minutes at 95°C, which will completely remove the CleanAmp™ modification from the oligonucleotide.
  4. Turbo should have less than 1% of the fully deprotected oligonucleotide while Precision should contain less than 2% of that contaminant. Precision should have less than 20% of the singly modified material, which elutes between the unmodified product and the doubly modified Precision oligonucleotide.
  5. Representative chromatograms of the CleanAmp™ primer sequences shown in Figure 2 are shown in Figures 3 and 4 on the following page.
Figure 3
Figure 3: RP HPLC analysis of a CleanAmp™ Turbo Primer
Figure 4
Figure 4: RP HPLC analysis of a CleanAmp™ Precision Primer

Product Information

10-1440
CleanAmp™-Pac-dA
10-1450
CleanAmp™-Ac-dC
10-1460
CleanAmp™-Pac-dG
10-1470
CleanAmp™-dT
Figure 1: structures Of CleanAmp™ Phosphoramidites

Hot Start PCR

Please also see : Hot Start PCR update: CleanAmp™ Primers and Purification of CleanAmp™ DNA Oligonucleotides (DMT-ON)