Applicable Products
LK2158 |
Quasar™ 570-CE Phosphoramidite |
LK2159 |
Quasar™ 670-CE Phosphoramidite |
LK2412 |
3'-Cyanine-3 SynBase™ CPG 1000/110 (Cyanine 540) |
LK2413 |
3'-Cyanine-5 SynBase™ CPG 1000/110 (Cyanine 650) |
LK2520 |
Cyanine-3-CE Phosphoramidite (Cyanine 540) |
LK2521 |
Cyanine-5-CE Phosphoramidite (Cyanine 650) |
Physical & Dilution Data
Dilution volumes (in ml) are for 0.1M solutions in dry acetonitrile (LK4050) with the exception of LK2521 where anhydrous alcohol free DCM is required. Adjust accordingly for other concentrations. For µmol pack sizes, products should be diluted as 100µmol/ml to achieve 0.1M, regardless of molecular weight. We suggest also using 3-4Å activated molecular sieves (10% v/v).
Item
|
Mol. Formula
|
Mol. Wt.
|
Unit Wt.
|
250mg
|
500mg
|
1g
|
LK2158 |
C44H65N5O4P |
759.00 |
622.74 |
3.29 |
6.59 |
13.18 |
LK2159 |
C46H67N5O4P |
758.04 |
648.78 |
3.18 |
6.37 |
12.74 |
LK2412 |
- |
- |
507.59 |
- |
- |
- |
LK2413 |
- |
- |
633.63 |
- |
- |
- |
LK2520 |
C58H70ClN4O4P |
953.64 |
507.59 |
2.62 |
5.24 |
10.49 |
LK2521 |
C60H72ClN4O4P |
979.68 |
533.63 |
2.55 |
5.10 |
10.21 |
Coupling
A 10min coupling time is recommended for all phosphoramidites. Due to the milder conditions required when deprotecting cyanine-labelled oligonucleotides, the use of fast or ultramild deprotection amidites (Ac-dC, Bz-dA, dmf-dG, iPrPac-dG, Pac-dA) is recommended. The use of 4A molecular seives is recommended.
Deprotection
As these reagents require no deprotection themselves, the oligonucleotide deprotection strategy employed is guided by the protective groups on the rest of the oligo. However, the mildest possible conditions are recommended, especially for Quasar™ 670 (LK2159) and Cyanine 5 (LK2521) which are susceptible to degradation at elevated temperatures.
Cleavage and deprotection: Do this in one step. Remove the resin from the column and place into a deprotection tube. Add ammonium hydroxide and allow to stand for 2-3hr at room temperature. The deprotection solution must be removed immediately after deprotection (see below).
Biosearch recommend deprotection of the Quasar dyes with ammonium hydroxide for 8hr at 25°C or for 1 hr at 60°C, however we have obtained good quality oligos with the above conditions, especially as elevated temperatures increase degradation.
Removing the deprotection solution: This must be done as soon as the deprotection step is complete. It is important not to heat the oligo to remove the deprotection solution since this will degrade the dye. Pass the solution through a G25 (NAP10 or equivalent) column. This will remove the basic solution and the sample is ready for purification. Some users pass the solution through two columns to be absolutely sure that the solution is now ~ neutral pH.
Spectral Data
|
Absorbance Max./nm
|
Emission Max./nm
|
Colour
|
Quasar™ 570 |
547 |
570 |
Yellow-Orange |
Quasar™ 670 |
644 |
670 |
Red |
Cyanine 3 |
546 |
563 |
Yellow-Red |
Cyanine 5 |
646 |
662 |
Violet-Far Red |
Storage & Stability
These products are light-sensitive and are susceptible to oxidation when left exposed to air and/or moisture. Store dry in a freezer, in a sealed dark or amber container at –20°C. Similarly, modified oligonucleotides must also be protected from exposure to light. The phosphoramidites are stable in solution under argon for 24h.