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Catalog Number: 10-1006-xx
Description: Etheno-dA-CE Phosphoramidite
| 5'-Dimethoxytrityl-etheno-deoxyAdenosine3'-[(2-cyanoethyl)- (N,N-diisopropyl)]-phosphoramidite | ||
| Formula: C42H48N7O6P | M.W.: 777.86 | F.W.: 337.23 |
Diluent: Anhydrous Acetonitrile |
| Coupling: No changes needed from standard method recommended by synthesizer manufacturer. The use of UltraMILD monomers are preferred. (Catalog Numbers: dA: 10-1601-xx, dC: 10-1015-xx, dG: 10-1621-xx, dT: 10-1030-xx). To avoid any exchange of the iPr-Pac group on the dG with acetyl, use the UltraMild Cap Mix A (40-4210-xx/ 40-4212-xx). |
| Deprotection: If UltraMILD reagents were used, deprotect in 0.05M Potassium Carbonate in Methanol for 4 hours at Room Temperature OR for 2 hours at hours in 30% Ammonium Hydroxide. If standard bases were used, deprotection in Ammonium Hydroxide at Room Temperature for 24-36 hours will give acceptable yields. |
| Storage: Refrigerated storage, maximum of 2-8°C, dry |
| Stability in Solution: 1-2days |
| Catalog Information | Material Safety Data Sheet |
EXTINCTION DATA
| Item | Nucleoside | λMax-1 | Emax-1 | λMax-2 | Emax-2 | E260 |
| (nm) | (ml/µmole) | nm | (ml/µmole) | (ml/µmole) | ||
| 10-1006 | Etheno-dA | 295 | 3.4 | 274 | 5.9 | 4.7 |
Literature Highlights
| Glen Report 6.1: ETHENO-DA AND ETHENO-A* - FLUORESCENT MONOMERS |
Frequently Asked Technical Question
QUESTION: Can Etheno-dA be substituted for dA in a sequencing or PCR primer . What is the best method for cleavage/deprotection of oligos containing Etheno-dA. What are the excitation/emmision wavelengths for oligos containing etheno-dA.?
RESPONSE:Due to the structure of the 1, N6 etheno structure etheno dA will not base-pair with T or dU. For this reason oligonucleotides with etheno-dA in the middle or 3'-end will not act as PCR or sequencing primers. However if located at or near the 5''-end etheno-dA can be incorporated one or more times and still act as an effective sequencing or PCR primer (1).
Etheno-dA is somewhat labile to NH4OH so it is best to use base labile protecting groups such as Pac dA, isopropyl-Pac-dG and Ac-dC and either deprotect with K2CO3:MeOH, 4 hr. at RT. or NH4OH 4 hr. at RT.. If using standard protecting groups use mild deprotection conditions (0.4 M Methanolic NaOH; MeOH:H2O, 4:1) 17 hr. at room temperature.
| Excitation | Emission | |
| Ribo etheno-A | 345 nm | 455 nm |
| Oligo-etheno-dA | 270 nm | 410 nm |
| 300 nm | 410 nm |
REFERENCE(S):
1. Srivastava, S.C., et.al., (1994), Nucleic Acids Research, 22, 1296-1304.
QUESTION: What are the relative extinction coefficients of 5'-Fluorescein, Hex and Tet etc.. at 260 nm and their Lambda max?
RESPONSE:Please see http://www.glenresearch.com/Technical/Extinctions.html
REFERENCE(S):
Oligonucleotide Properties Calculator; http://www.basic.northwestern.edu/biotools/oligocalc.html
QUESTION: What are the relative extinction coefficients of various dyes?
RESPONSE:Please see http://www.glenres.com/Technical/Extinctions.html#dyes
QUESTION: Does AMA or methylamine cause any degradation to fluorescein or fluorescein-type dyes such as FAM or FITC?
RESPONSE:Response: While AMA (Ammonium hydroxide/40% Methylamine 1:1 v/v) is considered compatible with fluorescein, the use of methylamine when deprotecting a Fluorescein-labeled oligo does lead to a small amount of degradation, which is characterized by a the appearance of a late-eluting peak by RP HPLC that shows no visible fluorescein absorbance. With standard deprotection conditions (AMA 10 minutes at 65 C) the amount of degradation is approximately 5%. The impurity is not detected with AMA at RT for 2 hours.
DILUTION/COUPLING DATA
The table below shows pack size data and, for solutions, dilutions and approximate couplings based on normal priming procedures. Please link for more detailed usage information with the various synthesizers.
| ABI 392/394 | |||||||||
| Cat.No. | Pack Size | Grams/ Pack | 0.1M Dil. (mL) | LV40 | LV200 | 40nm | 0.2µm | 1µm | 10µm |
| Approximate Number of Additions | |||||||||
| 10-1006-02 | 0.25grams | .25grams | 3.21 | 93.67 | 56.2 | 35.13 | 25.55 | 18.73 | 4.68 |
| 10-1006-90 | 100µmoles | .078grams | 1 | 20 | 12 | 7.5 | 5.45 | 4 | 1 |
| Expedite | |||||||||
| Cat.No. | Pack Size | Grams/ Pack | Dilution (mL) | Molarity | 50nm | 0.2µm | 1µm | 15µm | |
| Approximate Number of Additions | |||||||||
| 10-1006-02 | 0.25grams | .25grams | 4.8 | .07 | 89.6 | 56 | 40.73 | 5.6 | |
| 10-1006-90 | 100µmoles | .078grams | 1.5 | .07 | 23.6 | 14.75 | 10.73 | 1.48 | |
| Beckman | |||||||||
| Cat.No. | Pack Size | Grams/ Pack | Dilution (mL) | Molarity | 30nm | 200nm | 1000nm | ||
| Approximate Number of Additions | |||||||||
| 10-1006-02 | 0.25grams | .25grams | 4.8 | .07 | 91.2 | 57 | 41.45 | ||
| 10-1006-90 | 100µmoles | .078grams | 1.5 | .07 | 25.2 | 15.75 | 11.45 | ||
08/15/2012 | http://www.glenres.com/ProductFiles/10-1006.html