5' Phosphorylating Amidite II (O-DMT-2,2-di(ethoxycarbonyl)propan-1,3-diol)
货号：BNS-5009-100,BNS-5009-250
规格：100 µmol,250 mg
价格：703.5,2251
产品类型：核酸合成
品牌：LGC Biosearch Technologies
| Oligonucleotides having a 5'-phosphate group are valuable tools for many molecular biology applications including gene construction, cloning, mutagensis, and ligation chain reaction. Several methods have been developed to allow for the 5' phosphorylation of synthetic oligonucleotides. The most common is through the use of a 5'Phosphate Amidite (2-O-4,4'-dimethoxytrityl-2'-oxydiethane sulfonyl)-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite (BNS-5010). Unfortunately, the DMT group cannot be left on for DMT-ON purification due to the elimination reaction of the DMT and sulfonyl ethyl group during normal ammonium hydroxide deprotection and cleavage.
Phosphorylating Amidite II contains a DMT group that can be removed to determine the coupling efficiency of the amidite or it may be left on to allow for reverse phase cartridge purification. To determine the coupling efficiency, the DMT group is removed on the synthesizer. The side chain is eliminated, during ammonium hydroxide deprotection and cleavage, to yield a 5'Phosphate. For DMT-ON purification, the DMT group is removed with aqueous acid (3% TFA) and the side chain is eliminated after treatment with ammonium hydroxide. |
| 特性： |
| - Chemical Name: 3-(4,4'-dimethoxytrityloxy)-2,2-di(ethoxycarbonyl)propyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite - Formula: C39H53N2O9P - Molecular Weight: 722.8 - Appearance: colorless oil |
| 用途： |
| Synthesis conditions:Prior to dilution ensure that all product is at the bottom of the vial. Dilute to the recommended concentration and mix thoroughly in the sealed vial to ensure that all contents are dissolved.Dilution:100 µmol/mLCoupling:10-minute couplingDeprotection conditions:The deprotecting conditions for this amidite will be depend on the types of amidites used for the synthesis of the oligo. If using fast deprotecting amidites deprotect in concentrated NH4OH for 1 hour at 60 °C. If using standard amidites deprotect in concentrated NH4OH for 5 hours at 60 °C.The DMT group can be left on for cartridge purification. Below is a protocol using reversed-phase MicroPure column (MP-1602) for purification of oligonucleotides. Each MicroPure column will clean up to a 1µmol synthesis column.
1. Rinse column with MeCN (2 x 2 mL).
2. Activate column with 1N TEAA (3 x 2 mL)
3. Add Crude DMT-Oligo, 1mL of 1:1 solution (concentrated aqueous NH4OH/H2O). (Collect and reload solution twice)
4. Elute Failures with 3% NH4OH (3 x 2 mL).
5. Rinse column with water (3 x 2 mL).
6. Deblock DMT with 2.5 % TFA aqueous (3 x 2 mL). Wait 5 minutes between applications.
7. Rinse excess TFA with water. (3 x 2 mL; rinse until pH is neutral).
8. Insert collection tubes; elute purified oligo with 20% MeCN in water. (1 x 1.5 mL)
9. Dry sample in speed vac
10. To produce 5' phosphate, dissolve pellet in 0.5 mL of concentrated NH4OH. Leave in solution for 30 minutes at 60 °C.
11. Speedvac sample.
Preparation of Reagents:
Five different solutions need to be prepared for the purification process. To store these solutions use clean glass bottles with plastic lined caps.
1. Water (Use good quality sterile, deionized water. HPLC grade is best, especially if subsequent analyses will rely on HPLC methods.)
2. Acetonitrile (VWR, cat. number JT9018-3).
3. 20% acetonitrile in water. Dilute 20 mL of the same acetonitrile as in 2 above with 80 mL of the same water as in 1 above.
4. 1.0 N Triethylammonium acetate (TEAA). For this solution, place about 80 mL of water (from #1 above) in a clean bottle, and add exactly 5.7 mL of acetic acid (Aldrich cat. number 24, 285-3). Swirl the solution until completely mixed, and add exactly 13.9 mL of triethylamine (Aldrich cat. number 23, 962-3). Mix the solution until it is completely homogeneous - a small stir bar and magnetic stirrer may be used with good results. After complete mixing is achieved, the pH should be neutral, pH paper can be used for this (VWR cat. number 60775-702).
Add water to bring total volume to 100 mL. (Caution - wear eye protection and gloves when using concentrated acetic acid and triethylamine, concentrated acetic acid causes burns)
5. 2.5% Trifluoroacetic acid (TFA) in water. Carefully add 2.5 mL of TFA (Aldrich cat. number T6, 220-0) to 97.5 mL of water (#1 from above). Mix the solution until it is completely homogeneous.
(Caution - wear eye protection and gloves when using concentrated TFA)
6. 3.0% Ammonium Hydroxide in water. (Add 10.7 mL of 28% aqueous ammonia (Aldrich cat. number 22, 122-8) to 89.3 mL of water (from #1 above).) Mix the solution until it is completely homogeneous.
(Caution - wear eye protection and gloves when using concentrated ammonia)Image of cleaved and deprotected structure: The mass this product adds after conjugation and work-up (the additional mass seen by mass spectrometry) is:79.99 |
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