Training manual

Cartridge Purification

These are the instructions to follow for the purification of 5’ CPR II Modified Oligonucleotide. There are exceptions
depending on the modifications made on the oligonucleotide. Check with the scientist with regards to which
protocol to use.

Read the actual protocol: https://www.glenresearch.com/media/productattach/g/l/glen-pak_2.9_1 .

Materials Needed:

 1 Cartridge
 3 2mL Screwcap Tube (1 for Loading Error (LE), 1 for eluent 1 (E1), 1 for eluent 2 (E2))
 1 Small conical flask
 1 Needle
 1mL 100 mg/mL NaCl
 0.5mL ACN
 1mL 2M TEAA
 2mL Salt Wash
 2mL 4% TFA
 2mL deionized water
 1mL 50% ACN/water containing 0.5% NH3
 1mL concentrated NH4OH

Order of Chemical Used:

 1mL 100 mg/mL NaCl (Break into 0.5, 0.5)

 0.5mL ACN
 1mL 2M TEAA (Break into 0.5, 0.5)
 2mL Salt Wash (Break into 0.67, 0.66, 0.66)
 2mL 4% TFA (Break into 0.67, 0.66, 0.66)
 2mL deionized water (Break into 0.67, 0.66, 0.66)
 1mL 50% ACN/water containing 0.5% NH3 (For each, break into 0.5, 0.5)
 1mL concentrated NH4OH (For each, break into 0.5, 0.5)
Sample Preparation: in one note). Collect the liquid in the waste
container
1. Transfer the CPG to a 2mL Screwcap Tube and
7. Wash the cartridge with 2.01mL deionized
add 0.50mL NH4OH and 0.50mL methylamine.
water (break into 0.67, 0.67, 0.67). Collect
This will be the ‘sample’. (Do under a fume
the liquid in the small conical flask. (Flush
hood)
acid,
2. Put the sample in the thermomixer at ‘55°C’
8. Remove the needle from the cartridge. (The
for ‘2 hours’.
needle may still contain 4% TFA)
3. Remove the sample from the thermomixer
9. Put one of the screwcap tubes under the
and let it rest for 10 to 15 minutes. (This is to
cartridge and elute the oligo with 0.50mL 50%
prevent splashing due to pressure)
ACN/water containing 0.50% NH3. Do 2 times
4. Add 1mL 100 mg/mL NaCl into sample. (For
(1 to E1 1 to E2)
short oligonucleotide, put the sample back
into the thermomixer for 5 minutes at room
temperature/25°C) (For long oligonucleotide,
put the sample back into the thermomixer
for 5 minutes at 45°C) 10. Label this screwcap tube as ‘sample E1’.
5. After removing, let it rest to avoid splashing. 11. Repeat step 6 with another screwcap tube
and label this as ‘sample E2’.
Cartridge Preparation:
12. Add 0.50mL concentrated NH4OH to both
1. Attach the needle to cartridge. screwcap tubes. (Do under a fume hood)
2. Condition the cartridge with 0.50mL ACN. 13. Put ‘sample E1’ and ‘sample E2’ in the
Collect the liquid in a small conical flask. thermomixer at ‘30°C’ for ‘2 hours’.
3. Condition the cartridge with 1mL 2M TEAA 14. Put ‘sample E1’ and ‘sample E2’ in the -50°C
(break into 0.50, 0.50). fridge till they freeze.
4. Make a hole in the grey rubber stopper 15. After freezing, put ‘sample E1’ and ‘sample E2’
(septum) in the CentriVap to lyophilize the sample.
5. Push syringe into the hole and pressurize the
Things to take note:
liquid (1 drop/sec)
6. *WATER LEVEL CANNOT BE BELOW WHITE 1. If the temperature of the thermomixer is
FILTER. above/below the required temperature,
please allow it to drop/rise before placing the
Purification:
sample.
1. Label 3 tubes (Sample name, E1, E2, Loading 2. If there is insufficient time after deprotection,
error) put the sample in the 4°C fridge, without
2. Needle slightly below into the tube. adding 100 mg/mL NaCl.
3. Prepare another septum. 3. When applying pressure, apply it slowly to
4. Apply the whole 2.01mL sample (break into allow complete washing.
0.67, 0.67, 0.67) to cartridge. Collect the 4. Do not allow the cartridge to dry out.
eluent in a screwcap tube and label this as 5. If the time is insufficient to complete
‘sample LE’. (Take the CPG) (ALL INTO ammonium incubation, set the thermomixer
LOADING ERROR) at ‘25°C’ till the next day’s morning.
5. Wash the cartridge with 2.01mL Salt Wash 6. You may use liquid nitrogen to freeze the
(5%ACN) (break into 0.67, 0.67, 0.67). [Add sample. Handle with care.
salt wash (ACN) into original sample tube]
{ First salt wash back into the sample tube}
[Second wash into waste][3rd wash waste
container]
6. Apply 2.01mL 4% TFA (break into 0.67, 0.67,
0.67, 0.67) and allow the acid to flow slowly
for 2 to 3*** minutes for short or 3 to 4
minutes*** for long (Orange band MUST be
visible for oligo with DMT groups note down
Preparation of 1L 1M TEAA buffer solution Things to take note:

1. Clean the measuring cylinders and a glass 1. This preparation MUST be done in a fume
bottle with tap water, soap and then acetone. hood.
Flush them with air after washing with 2. Add acetic acid and triethylamine slowly.
acetone. 3. Wait for the buffer to rise to approximately
2. Prepare an ice bath. room temperature before measuring the pH.
3. Measure out 799mL water and pour 4. For pH meter, please ensure it is cleaned with
approximately 300mL into the glass bottle. DI water at the beginning and the ending of
(This is to allow easier mixing) use.
4. Put the bottle into the ice bath. 5. Place the cover back and ensure that the tip
5. Measure out 144mL triethylamine and 57mL (where it is coloured) is submerged in the KCl.
acetic acid. 6. Please set the vacuum correctly. The buffer
6. While the glass bottle is in the ice bath, add should be filter as fast droplets or a slow
the acetic acid in small amounts until all the stream of water.
acetic acid is added to the water. (Do not add
large amount of acetic acid to the water as
the reaction is exothermic) (Note that there
may be white fume present and it is normal)
7. After all the acetic acid is added, add 1mL of
triethylamine drop by drop slowly with a
micropipette 10 times. (This is to give a brief
understanding of how the reaction will be
like) (You can add small amounts after
adding these 10 1mL)
8. Since triethylamine does not easily dissolve in
water and needs to react with acetic acid,
shake the bottle while it is in the ice bath until
the triethylamine dissolve.
a. Ensure that the bottle is cold before
adding triethylamine
b. The liquid should turn colourless
when no air bubbles are seen
c. Note that there may be white fume
present and white precipitate formed
d. If white precipitate has formed, this is
a sign of adding the triethylamine too
fast or too much at once. This does
not affect the preparation
9. Use a pH meter to determine the pH of the
solution. (If acidic, add 1-2 drops of
triethylamine and shake the solution) (If
alkaline, add 1-2 drops of acetic acid and
shake the solution)
10. Repeat step 9 until the pH is in the range of
7.0-7.4.
11. Use a vacuum filter on the prepared mixture
and collected the filtrate.
12. Label the bottle as ‘1M TEAA pH ___’, where
the pH is the buffer you prepared.
Preparation of 1L 1M HAA buffer solution Things to take note:

13. Clean the measuring cylinders and a glass 1. This preparation MUST be done in a
bottle with tap water, soap and then acetone. fumehood.
Flush them with air after washing with 2. Add acetic acid and hexylamine slowly.
acetone. 3. Wait for the buffer to rise to approximately
1. Prepare an ice bath. room temperature before measuring the pH.
2. Measure out 812mL water and pour 4. For pH meter, please ensure it is cleaned with
approximately 300mL into the glass bottle. DI water at the beginning and the ending of
(This is to allow easier mixing and use.
adjustment of pH) 5. Place the cover back and ensure that the tip
3. Put the bottle into the ice bath. (where it is coloured) is submerged in the KCl.
4. Measure out 131mL hexylamine and 57mL 6. Please set the vacuum correctly. The buffer
acetic acid. should be filter as fast droplets or a slow
5. While the glass bottle is in the ice bath, add stream of water.
the acetic acid in small amounts until all the 7. molar = mol/litre
acetic acid is added to the water. (Do not add 8. mol = gram/MW
large amount of acetic acid to the water as
the reaction is exothermic) (Note that there
may be white fume present and it is normal)
6. After all the acetic acid is added, add 1mL of
hexylamine drop by drop slowly with a
micropipette 10 times. (This is to give a brief
understanding of how the reaction will be
like) (You can add small amounts after less or
no fume seen after adding 1ml)
7. Since hexylamine does not easily dissolve in
water and needs to react with acetic acid,
shake the bottle while it is in the ice bath until
the triethylamine dissolve.
a. Ensure that the bottle is cold before
adding hexylamine
b. Note that there may be white fume
present and white precipitate formed
c. If white precipitate has formed, this is
a sign of adding the hexylamine too
fast or too much at once. This does
not affect the preparation
8. Use a pH meter to determine the pH of the
solution. (If acidic, add hexylamine and shake
the solution) (If alkaline, add acetic acid and
shake the solution)
9. Repeat step 9 until the pH is in the range of
7.0-7.4.
10. Use a vacuum filter on the prepared mixture
and collected the filtrate.
11. Label the bottle as ‘1M HAA pH ___’, where
the pH is the buffer you prepared.
Protocol for Deprotection and DMT-On Glen - PakTM
Purification

Sample Preparation:
Desalting DMT-off DNA Oligos:
1. Make a fresh solution of 0.4M NaOH (MW is
1. Load the solution containing the
39.997 g/mol) in MeOH/water 4:1 (v/v).
oligonucleotide onto the cartridge slowly.
(Always prepare it freshly and slightly more
(Collect the effluent as ‘LE 1’ in another 15mL
than required) (Do under a fume hood)
falcon tube)
2. If it is desired to eliminate the cyanoethyl
2. Flush the cartridge with 1mL 0.1M TEAA
protecting groups on the phosphate
twice. (Collect as ‘LE 2’)
backbone, treat the column with 3mL 10%
3. Flush the cartridge with 1mL deionized water
diethylamine (DEA) in ACN for 2 minutes,
twice. This wash removes the salts from the
pushing the solution back and forth
cartridge. (Also collect it in ‘LE 2’)
occasionally. Rinse with ACN and air dry the
4. Elute the desalted oligonucleotide by flushing
CPG. (Usually done by scientist)
the cartridge with 1mL 10% ACN/water and
3. Transfer the CPG to a 2mL Screwcap Tube and
label as ‘E1’. The product should elute fully in
add 1 mL 0.4M NaOH in MeOH/water 4:1
1mL while leaving behind organics such as
(v/v).
benzamide on the column matrix.
4. Put it in the thermomixer and allow it to react
5. Elute a second time with 1mL 50% ACN/Water
for at least 17 hours at room temperature. (If
and label as ‘E2’. This allows maximum
the temperature is above/below the
recovery of material but contains small
required temperature, please allow it to
molecule impurity.
drop/rise before placing the sample into the
6. Put ‘E1’ and ‘E2’ in the -80°C fridge for half to
thermomixer)
one hour till they freeze.
5. Briefly sonicate the Screwcap Tube to break
7. Put ‘E1’ and ‘E2’ in the CentriVap overnight to
up the CPG.
lyophilize the sample.
6. Vortex and spin down in a centrifuge.
7. Pipette off the supernatant and transfer to a Things to take note:
15mL falcon tube. (Try not to take the CPG)
8. Rinse the CPG with 250µL water. 1. When applying pressure, apply it slowly to
9. Vortex and spin down in a centrifuge. allow complete washing.
10. Pipette the 250µL water and combine with 2. Do not allow the cartridge to dry out. Left
the cleaved oligo. (Try not to take the CPG) some behind (except ‘E2’).
11. Dilute to 10mL with 100 mg/mL NaCl in water. 3. Transfer the liquid using a micropipette
12. Vortex and spin down in a centrifuge. slowly. Do not rush.
4. For ‘1.’ in ‘Desalting Procedure Crude
Cartridge Preparation: Deprotected DNA Oligos:’, do it very slowly.
5. When adding 1mL liquid into the 1mL syringe,
1. Attach the needle to cartridge.
the liquid may not drop down. Hence, try to
2. Condition the cartridge with 0.5mL ACN.
cover it with the micropipette tip to apply
Collect the liquid in the small conical flask.
pressure for the liquid to drop down.
3. Condition the cartridge with 1mL 2M TEAA
(break into 0.50, 0.50). Collect the liquid in
the small conical flask.
Protocol for Deprotection and DMT-On Glen - PakTM Protocol for Deprotection and DMT-On Glen - PakTM
Purification Purification

Sample Preparation: Desalting Procedure Crude Deprotected DNA Oligos:

1. Make a fresh solution of 0.4M NaOH (Molar 1. Apply the whole 2.01mL sample (break into
mass is 39.997 g/mol) in MeOH/water 4:1 0.67, 0.67, 0.67) to cartridge. Collect the
(v/v). (Always prepare it freshly and slightly eluent in a screwcap tube and label this as
more than required) (Do under a fume hood) ‘sample LE’. (Take the CPG)
2. If it is desired to eliminate the cyanoethyl 2. Wash the cartridge with 2.01mL Salt Wash
protecting groups on the phosphate (break into 0.67, 0.67, 0.67). (For the second
backbone, treat the column with 3mL 10% wash, collect it in the ‘sample’ tube to rinse
diethylamine (DEA) in ACN for 2 minutes, the tube to collect the oligonucleotides that
pushing the solution back and forth are left behind. Wash the cartridge with this
occasionally. Rinse with ACN and air dry the 0.67mL for the third wash)
CPG. (Done by scientist) 3. Apply 2.01mL 4% TFA (break into 0.67, 0.67,
3. Transfer the CPG to a 2mL Screwcap Tube and 0.67) and allow the acid to flow slowly for 2 to
add 1 mL 0.4M NaOH in MeOH/water 4:1 3 minutes for short or 3 to 4 minutes for long
(v/v). (Orange band MUST be visible for oligo with
4. Put it in the thermomixer and allow it to react DMT groups). Collect the liquid in the small
for at least 17 hours at room temperature. (If conical flask.
the temperature is above the required 4. Wash the cartridge with 2.01mL deionized
temperature, please allow it to drop before water (break into 0.67, 0.67, 0.67). Collect
placing the sample into the thermomixer) the liquid in the small conical flask.
5. Briefly sonicate the Screwcap Tube to break 5. Remove the needle from the cartridge. (The
up the CPG. needle may still contain 4% TFA)
6. Vortex and spin down in a centrifuge. 6. Put one of the screwcap tubes under the
7. Pipette off the supernatant and transfer to a cartridge and elute the oligo with 0.50mL 50%
15mL falcon tube. ACN/water containing 0.50% NH3.
8. Rinse the CPG with 250µL water. 7. Label this screwcap tube as ‘sample E1’.
9. Vortex and spin down in a centrifuge. 8. Repeat step 6 with another screwcap tube
10. Pipette the 250µL water and combine with and label this as ‘sample E2’.
the cleaved oligo. 9. Add 0.50mL concentrated NH4OH to both
11. Dilute to 10mL with 100 mg/mL NaCl in water. screwcap tubes. (Do under a fume hood)
12. Vortex and spin down in a centrifuge. 10. Put ‘sample E1’ and ‘sample E2’ in the
thermomixer at ‘30°C’ for ‘2 hours’.
Cartridge Preparation:
11. Put ‘sample E1’ and ‘sample E2’ in the -50°C
1. Attach the needle to cartridge. fridge till they freeze.
2. Condition the cartridge with 0.5mL ACN. 12. After freezing, put ‘sample E1’ and ‘sample E2’
Collect the liquid in the small conical flask. in the CentriVap to lyophilize the sample.
3. Condition the cartridge with 1mL 2M TEAA
(break into 0.50, 0.50). Collect the liquid in
the small conical flask.