此頁面上的某些內容不提供英文版本,將默認以當前可用的語言顯示。
Conjugation of Alkyne-modified Oligonucleotides with Dye Azides
Click chemistry buffer is intended for post-synthetic conjugation of oligonucleotides containing alkyne.
Oligonucleotides with terminal alkyne can be synthesized using alkyne
phosphoramidite, or you can order synthesis of modified oligonucleotides on our website.
Azide is conjugated to terminal alkyne
of a modified oligonucleotide, resulting in a five-membered heterocycle. Both groups (azide and
alkyne) are extremely rarely found in natural biomolecules, so the reaction is highly specific and
effective to handle various tasks.
The reaction proceeds in the presence of copper (I) at neutral pH. Catalytic buffer contains
copper (II), triethylammonium acetate pH 7 and DMSO. It is recommended to use a freshly
prepared solution of ascorbic acid to reduce copper (II).
For this
reaction, you will need alkyne-modified oligonucleotide, dye azide, click chemistry
buffer, and ascorbic acid. You can order all the reagents online on our
website.
Protocol
We recommend the following protocol for conjugation of alkyne-containing
oligonucleotides with dye azides:
- Determine total reaction volume based on the amount of oligonucleotide to be used:
Amount of oligonucleotide |
Total reaction volume, µL |
4 to 20 nmol |
100 |
20 to 40 nmol |
200 |
40 to 80 nmol |
400 |
80 to 600 nmol |
600 |
- Calculate volumes of the reagents for the labeling reaction using the table below:
Reagent |
Volume, µL |
Concentration of stock solution |
Dye azide |
(amount of oligonucleotide [nmol]) × 0.15 |
10 mM in DMSO |
Click chemistry buffer |
(total reaction volume [µL]) × 0.67 |
1.5х |
Activator (ascorbic acid) |
(total reaction volume [µL]) × 0.02 |
50 mM in water |
Water (for oligonucleotide dissolution) |
(total reaction volume [µL] - volume of dye azide solution [µL] -
volume of buffer [µL] - volume of activator solution [µL])
|
— |
- Prepare stock solution of dye azide (10 mM in DMSO) and activator (ascorbic
acid, 50 mM in water).
Bear in mind that ascorbic acid is readily oxidizable in air. Use only a freshly prepared
solution of activator (the solution is stable within 1 day). To prepare stock solution,
dissolve 10 mg of ascorbic acid in 1.1 mL of water.
- Dissolve oligonucleotide in the calculated volume of water in a 2-mL plastic tube.
- Add click chemistry buffer and vortex.
- Add the calculated volume of stock solution of dye azide and vortex again.
- (recommended). Degas the mixture to remove oxygen. To do so, connect a disposable
pipette tip to a plastic or silicone tubing connected to the pressure regulator of a gas
cylinder with inert gas (argon, nitrogen, or helium). Turn on very weak gas flow and put the tip down
in the tube so that it can be 3–10 mm higher than the liquid level avoiding
touching the liquid and tube walls. The gas flow should make a swirl in the liquid without spattering it.
Keep the tip in this position for 10–20 seconds.
If several labeling reactions are run simultaneously, a SpeedVac-type system can be used for
degassing. To do so, place the tubes in the system, turn on rotation, turn on vacuum
for 30–40 s, then turn off vacuum while feeding inert gas to the input of the system.
- Add activator solution (ascorbic acid), then purge the tube with inert gas for a few seconds and
close it.
- Vortex the solution. If a precipitate forms during the reaction, warm the tube up in hot
water (70–95 °C) until the precipitate dissolves and vortex the solution.
- Allow the mixture to stand at room temperature for 8–16 h.
- Add 2M solution of lithium perchlorate (1 volume per 5 volumes of the reaction
mixture), vortex the solution, and add extra pure acetone to 2 mL.
- Shake the tube and allow it to stand for 20 min at −20 °C.
- Separate the precipitate by centrifuging at 10,000 RPM for 10 min. Discard the
supernatant.
- Add 1 mL of acetone to the precipitate. Shake the tube several times and separate the precipitate
by centrifuging at 10,000 RPM for 10 min. Discard the supernatant.
- Allow the precipitate to dry at room temperature in the open tube for 1 h or place the
tube in the heating block at 65 °C for 10 min.
- Dissolve the precipitate in water and purify the target product by HPLC.
相關的產品
Cyanine3.5 疊氮化物,用於通過點擊化學進行生物分子標記。
Found better price?
Let us know
and we will propose the way forward!
用於 сlick сhemistry 的 Cyanine3 螢光染料的疊氮衍生物。
Found better price?
Let us know
and we will propose the way forward!
用於點擊化學的遠紅/近紅外熒光 Cyanine5.5 染料衍生物。
Found better price?
Let us know
and we will propose the way forward!
Cyanine5 疊氮化物是一種用 Cyanine5(一種流行的螢光染料)進行點擊化學標記的試劑。
Found better price?
Let us know
and we will propose the way forward!
用於點擊化學的長波長近紅外螢光染料疊氮化物。
Found better price?
Let us know
and we will propose the way forward!
用於點擊化學標記的近紅外 (NIR) 螢光染料。
Found better price?
Let us know
and we will propose the way forward!
FAM(熒光素)疊氮化物,純 5-異構體,用於連結化學標記,。
Found better price?
Let us know
and we will propose the way forward!
FAM(螢光素)疊氮化物,6-異構體,是一種常用於點擊化學的螢光染料疊氮化物。
Found better price?
Let us know
and we will propose the way forward!
JOE 是一種氯代螢光素染料,常用於寡核苷酸的 HEX 通道。它的疏水性比 HEX 弱。是一種疊氮衍生物,適用於點擊化學。
Found better price?
Let us know
and we will propose the way forward!
綠色-黃色發射 HEX 染料的疊氮化物,通過 [3+2]-偶極環加成與寡核苷酸偶合。
Found better price?
Let us know
and we will propose the way forward!
即用型催化緩衝液含有銅(II) 和 TBTA 配體。適用於核酸和小分子的點擊化學修飾。
Found better price?
Let us know
and we will propose the way forward!
物品數量不正確.