Glen-Pak Purification of 5umol Scale Reverse-DNA
Materials | Quantity |
Glen-Pak DNA Purification Cartridge 3g | 1 |
Vacuum Manifold with Pressure Gauge (inHg) | 1 |
VWR Microcentrifuge | 1 |
VWR Digital Vortex Mixer | 1 |
Thermo LabQuake Test Tube Rack | 1 |
UV/Vis Spectrophotometer | 1 |
50mL Conical Vials | 2 |
Countdown Timer | 1 |
Ammonium Hydroxide | 10mL |
100mg/mL Sodium Chloride/Water | 10mL |
HPLC-Grade Acetonitrile (ACN) | 20mL |
2M Triethylammonium Acetate (TEAA) | 20mL |
10% Acetonitrile in 100mg/mL Sodium Chloride (10% ACN) | 20mL |
2% Trifluoroacetic Acid/Water (2% TFA) | 20mL |
Distilled Water | 30mL |
0.5% Ammonium Hydroxide in 1:1 Acetonitrile/Water Solution (Elution Wash) | 20mL |
Procedure
DNA-Resin Cleavage
- Add 800uL of Ammonium Hydroxide to the 5umol vial of DNA and centrifuge for 30 sec.
- Repeat Step 1 and vortex well until Ammonium Hydroxide and DNA form a clear solution.
- Set DNA to spin on the Thermo LabQuake rack for 2 hours.
- Spin the vial in the centrifuge until the solution is concentrated at the bottom.
- Increase the volume up to 10mL with Ammonium Hydroxide.
- Add 10mL of 100mg/mL Sodium Chloride to each vial for a final volume of 20mL.
DNA Purification
- Attach the male end of the Glen-Pak cartridge to the female luer port on the manifold.
- Condition the cartridge with 2 aliquots of 10mL of 100% ACN, followed by 2 aliquots of 10mL of 2M TEAA.
- Load the DNA in 2 aliquots of 10mL each.
- Wash the cartridge with 2 aliquots of 10mL of 10% ACN solution.
- Wash the cartridge with 2 aliquots of 10mL of 2% TFA solution.
- Rinse with 2 aliquots of 10mL of distilled water.
- Elute the contents of the cartridge with 2 aliquots of 10mL of Elution Wash. Collect the elute in the 50mL conical vial.
All wash stages in the ‘DNA Purification’ Procedure are summarized in the table below with accompanying recommendations for wash times and pressures. Please note that time constraints are more important than pressure constraints.
Wash Step | Volume (mL) | Time (min) | Pressure (inHg) |
Condition [ACN] | 10 x2 | 4 | 2 |
Condition [2M TEAA] | 10 x2 | 8 | 5-6 |
DNA Load | 10 x2 | 10 | 4-5 |
10% ACN | 10 x2 | 10 | 4-5 |
2% TFA | 10 x2 | 5 | 5-6 |
Distilled Water | 10 x2 | 10 | 5 |
Elution Wash | 10 x2 | 10 | 4.5 |
Quality Control
1. Add 3mL of water to a new 50mL conical vial.
2. Add 40uL of purified DNA product to the vial.
3. Vortex the vial well and centrifuge it for 30 sec.
4. Tare the UV/Vis spectrophotometer with 100uL of water at the 260nm wavelength.
5. Measure the absorbance of100uL of the diluted DNA product at 260nm. The desired absorbance value should fall within the range 0.4 ≤ absorbance ≤ 0.6. Perform a Beer-Lambert calculation to obtain the final yield of the DNA.
6. Determine the purity of the DNA by obtaining a plot of Absorbance vs. Wavelength with 210-300nm wavelengths. Pure DNA will exhibit a local maximum value at 260nm.