Инструкции по использованию колонок/пластин для обессоливания C18
Product Introduction
C18 desalination columns/plates are filled with a C18 silica gel matrix, whose functional group, octadecyl, has high hydrophobicity and excellent retention properties for non-polar compounds. C18 desalination columns/plates can adsorb primer/oligo/DNA molecules with DMT without salt retention, resulting in high purity primer/oligo/DNA products.
Selection Guide
BM Life Sciences offers C18 desalination columns/plates in a variety of sizes, and you can choose the right desalting column based on the primer/oligo concentration. For batch processing, you can choose a 96-well desalting plate.
Product Number | norm | Applicable oligonucleotide specifications | Reference steps |
SPEC18W150 | 50mg/1mL | 10nmol - 200nmol | Step 5 |
SPEC18W3100 | 100mg/3mL | 10nmol - 600nmol | Step 1 |
SPEC18W3300 | 300mg/3mL | 10nmol - 2 .opmol | Step 2 |
SPEC18W61000 | 1g/6mL | 1 umol - 6 umol | Step 3 |
SPEC18W9630 | 30mg/96 well plate | 5nmol - 100nmol | Step 4 |
SPEC18W9650 | 50mg/96 well plate | 10nmol - 200nmol | Step 4 |
SPE15C18W9680 | 80mg/96-well plate(1.5ml 96-well plate) | 10nmol - 500nmol | Step 5 |
SPE23C18W9680 | 80mg/96 well plate(2.3ml 96 well plate) | 10nmol - 500nmol | Step 5 |
Note
C18 Cleaning Column/Plate can also desalting raw product and primer/oligo after HPLC and PAGE purification, see step 5 for details.
Reagent
Acetonitrile Chromatographic Grade
0.1 M TEA, PH7.0
0.5 M Nacl
Flushing solution (5% acetonitrile dissolved in 0.5 M Nacl)
3% trifluoroacetic acid (TFA)
Deionized water
20% acetonitrile
TE buffer: 10 mmol/L Tris-cla, 1 mmol/L EDTA, PH 8.0
Step 1
Sample Preparation
Add 0.5 M NacI to the oligonucleotide sample to a volume of 2 mL.
Activating the desalination column
Activation: 1 mL of acetonitrile was used to activate the column.
Equilibroving: Balance column with 1 mL of 0.1 M TEAA solution (PH7.0).
Cleaning process
1. Pass 2 mL of oligonucleotide-containing solution through a desalination column.
2. Rinse the column 2 times with 1 mL of flushing solution to remove unsuccessful sequences.
3. Rinse the column 2 times with 1 ml of deionized water to remove salts.
4. Rinse the column 2 times with 1 ml of 3% trifluoroacetic acid to remove DMT and observe that the adsorbed layer turns orange-red; and
5. Rinse the desalting column twice with 1 mL of deionized water to remove trifluoroacetic acid and residual salts.
6. Elut 1ml of 20% acetonitrile and assemble.
Step 2
Sample Preparation
Add 0.5 M NacI to the oligonucleotide sample to a volume of 4 mL.
Activating the desalination column
Activation: 2 mL of acetonitrile was used to activate the column.
Equilibroving: Balance a 2 mL column with 0.1 M TEAA (PH 7.0).
Cleaning process
1. Pass 4 mL of oligonucleotide-containing solution through a desalination column.
2. Rinse the column 2 times with 2 ml of flushing solution to remove unsuccessful sequences.
3. Rinse the column 2 times with 2 ml of deionized water to remove salts ;)
4. Rinse the column 2 times with 2ml of 3% trifluoroacetic acid to remove DMT, the adsorption layer has turned orange-red.
5. Rinse the column with demineralized water twice with 2 mL of deionized water to remove trifluoroacetic acid and residual salts.
6. Elut 2ml of 20% acetonitrile and assemble.
Step 3
Sample Preparation
Add 0.5 M Nac to the oligonucleotide sample to a volume of 10 mL.
Activating the desalination column
Activation: 5 mL of acetonitrile was used to activate the column.
Equilibroving: The column was balanced with 10 mL of 0.1 m TEAA (PH 7.0).
Cleaning process
1. 10 mL of solution containing oligonucleotides was passed through a desalination column.
2. The column was washed twice with 5 mL of flushing solution to remove unsuccessful sequences.
3. Rinse the column twice with 5 mL of deionized water to remove salts.
4. Wash the column twice with 5 mL of 3% trifluoroacetic acid to remove DMT and notice that the adsorption layer has turned orange-red.
5. Rinse the column twice with 5ml of deionized water to remove trifluoroacetic acid and salt residues.
6. Elute 5 ml of 20% acetonitrile and collect.
Step 4
Sample preparation
Add 0.5 M Nacl to the oligonucleotide sample to a volume of 0.5 mL.
96-well plate activation
Activation: 0.5 mL of acetonitrile was passed through each well of a 96-well plate.
Equilibrium: Equilibrate a 96-well plate with 0.5 mL of 0.1 M TEAA solution (pH 7.0).
Cleaning procedure
1. Pass 0.5 mL of oligonucleotide-containing solution through a 96-well plate.
2. Rinse the 96-well plate 2 times with 0.5 mL of wash solution to remove bad sequences.
3. Rinse the 96-well plate 2 times with 0.5 mL of deionized water to remove salts.
4. Wash the 96-well plate 2 times with 0.5ml of 3% trifluoroacetic acid to remove DMT, and observe that the adsorption layer turns orange-red; and
5. Rinse the 96-well plate twice with 0.5 mL of deionized water to remove trifluoroacetic acid and salt residue.
6. Elut 0.5ml of 20% acetonitrile and collect the material.
Step 5
Sample preparation
Add 0.5 M Nacl to the oligonucleotide sample to a volume of 1.2/2 mL.
96-well plate activation
Activation: Skip 1-4 ml 0. 5 mL of acetonitrile through each well of a 96-well plate.
Equilibroving: Balance a 96-well plate with 1-4 mL of 0.5 mL of 0.1 M TEAA solution (PH 7.0).
Cleaning process
1. Pass 0.5-2 mL of oligonucleotide-containing solution through a 96-well plate.
2. Rinse the 96-well plate 2 times with 1/2 mL of wash solution to remove the unfortunate sequences ;)
3. Rinse the 96-well plate twice with 1/2 mL of deionized water to remove salts.
4. 2 times wash the 96-well plate with 1/2ml of 3% trifluoroacetic acid to remove DMT, and observe that the adsorption layer turns orange-red;
5. Rinse the 96-well plate with 1/2 mL of deionized water 2 times to remove trifluoroacetic acid and salt residue.
6. Elut 1/2ml of 20% acetonitrile and collect the material.
Step 6
Reagent
Acetonitrile Chromatographic Grade
0.05 M TEA, PH 7.0
2 M TEA, PH 7.0
Deionized water (for desalination and purification of RNA molecules without RNase) 60% acetonitrile (dissolved in water containing 0.5% NH4OH)
20% acetonitrile (to desalinate DNA or RNA molecules after deprotection)
TE buffer: 10 mmol/L Tris-cla, 1 mmol/L EDTA, PH 8.0
Sample Preparation
HPLC-purified oligo: Add an appropriate amount of 0.1 M TEAA to the HPLC-purified oligos so that the final acetonitrile concentration is ≤5%. Purified oligos: Dissolve the oligonucleotide-containing gel fragments in 0.05 M TEAA, remove the gel fragments, dissolve the oligo in at least 2 mL of 0.1 M TEAA.
Oligos dissolved in NH4OH or AMA: Evaporate until dry and dissolve in 2 mL of 0.1 M TEAA.
Activating the Desalination Pump
Activation: 1 mL of acetonitrile was used to activate the column.
Equilibroving: Balance a 1 mL 2 M TEAA column (pH 7.0).
Oligos desalination procedure for HPLC purification
1. Pass the solution containing oligonucleotides through the desalination column.
2. Rinse the desalination column twice with 1 mL of deionized water to remove the salts.
3. Elut through a column of 1 ml of 60% acetonitrile and collect.
Oligos desalting procedure purified by the PAAG method
1. Pass the solution containing oligonucleotides through the desalination column.
2. Rinse the desalination column twice with 1 mL of deionized water to remove the salts.
3. Elut through a column of 1 ml of 60% acetonitrile and collect.
Procedure for desalting DNA oligons after synthesis
1. Pass the solution containing oligonucleotides through the desalination column; and
2. Rinse the 1 mL 0.1M TEAA column twice to remove unsuccessful sequences.
3. Rinse the desalination column twice with 1ml of deionized water to remove the salts.
4. Pass the column through 1 ml of 20% acetonitrile and assemble.
RNA oligos desalting procedure after synthesis
1. Pass the solution containing oligonucleotides through the desalination column.
2. Rinse the column with 2 ml 0. 1 M TEAA.
3. Rinse the column with 2 mL of RNase-free water.
4. Pass 2ml of 20% acetonitrile (RNase-free water) through a column, elut and collect. Oligonucleotide Storage
Oligonucleotides dissolved in 20% acetonitrile and stored at 4°C prevent the growth of microorganisms and are suitable for short-term storage.
Oligonucleotides in a 20% acetonitrile solution (or dissolved in an alkaline buffer such as TE) can be stored for a long time at temperatures as low as -20 °C.
Oligonucleotides in the form of lyophilized powder can be stored for a long time.
Warning
Используемые количества реагентов являются лишь рекомендациями и должны быть оптимизированы в каждом конкретном случае.
The quantities of reagents used are only recommendations and should be optimized on a case-by-case basis.
The desalination column must undergo sufficient activation equilibrium to elute the residues on the column/plate and improve the binding capacity of the column/desalination plate. Sampling of synthesized oligonucleotides should be performed slowly to prevent CPG powder from entering the column/plate for cleaning and cross-contamination.
C18 desalination columns/wafers use silica matrix, the applicable PH range is 2-7, PH > 7.5 easily dissolves silica matrix, PH < 2 cracking of silica ester bonds, which affects the adsorption efficiency.
When using TFA to desalting DMT, pay attention to the time, too long will have the effect of removing the purine.
Be Attached
B&M Universal CPG Frits Synthesis Columns Rapid Desalting and Purification Technology
B&M Universal CPG Frits Synthesis Columns Rapid Desalination and Purification Technology:
B&M Universal CPG Frits are made of imported ultra-high molecular weight ultra-pure polyethylene and imported CPG powder, processed by special technology, have a good synthesis effect in the synthesis column, low mutation rate, and reliable product quality.
After the synthesis is completed in the Dr.Oligo/Mermade series synthesizer and ammonia is dissolved, Oligo can be eluted directly to the synthesis column, and after dosing and draining, it can be used for routine PCR reaction directly without additional purification steps, which is convenient to operate, save cost and faster delivery!
The specific steps of the operation are as follows:
1, Synthesis B&M Universal CPG Frits synthetic columns/plates/s 400ul, 75% acetonitrile ultrapure water mixture washed twice to remove synthetic residual reagents;
2, B&M Universal CPG Frits Synthetic Ammonia Ammolysis Columns/Plates, 90°C, 80PSI, Ammonia Ammolysis 2-2.5 h;
3, 100% acetonitrile 400L wash synthetic column/plate, twice;
4、90%Acetonitrile Ultrapure Water Blend 400ul Washing Synthetic Column/Plate, Twice, Pumping Dry;
5、Appropriate volume of eluent for eluting primers on synthetic column/plate, the eluent can be ultrapure water, can be 3% triethylamine ultrapure water mixture, also can be about 20% acetonitrile ultrapure water mixture.
6、 Elution solution can be measured, dispensed, drained, packaged and shipped.