C3 Spacer Amidite (DMT-1,3-Propanediol)
货号：BNS-5041-100,BNS-5041-250
规格：100 µmol,250 mg
价格：914.55,2955
产品类型：核酸合成
品牌：LGC Biosearch Technologies
| Thiol Modifier C6-S-S Amidite is used to functionalize the 5' terminus of an oligonucleotide with a thiol group. Thiol modifications have become significant in the production of diagnostic probes. Thiol functionalization allows attachment to fluorescent dyes, maleimide compounds or conjugation to proteins through disulfide linkages. The DMT group can be used as a handle for RP purification and later removed with 100 mM DTT. |
| 特性： |
| - Chemical Name: 1-O-Dimethoxytrityl-hexyl-disulfide, 1'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite- Formula: C42H61N2O5P S2- Molecular Weight: 769.05- 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. If labeling the 5’ terminus, the DMT group may be left on after the synthesis for Reversed-phase Cartridge Purification.Dilution:100 µmol/mLCoupling:2-minute couplingDeprotection conditions:For DMT-OFF Synthesis (5' modification):
For the DMT OFF oligonulceotide, cleave and deprotect using NH4OH solution for 2 hours at 60 °C if using fast deprotecting amidites or 5 hours at 60°C for standard amidites. To reduce the disulfide linkage to generate a 5'thiol modified oligo, dry down sample then add 0.05M DTT in water. Heat sample for 5 hours, dry the sample down and reconstitute in water. The sample may need to be filtered before HPLC analysis.
For DMT-ON Synthesis (5' modification):
For the DMT ON oligonucleotide cleave and deprotect the oligo in concentrated NH4OH for 5 hours at 60 °C, for fast deprotecting amidites cleave and deprotect 2 housr at 60 °C. Purify the DMT ON oligonuleotide using a SuperPure Plus purification column (<200 nmol scale synthesis, see protocol below). Reduce the disulfide bond using 100 mM DTT in 0.2 M phosphate buffer pH 8.5 for 30 minutes at room temperature. Elute the 5' Thiol oligonucleotide and desalt using a G-25 or NAPS column to generate a purified 5' Thiol oligonucleotide.Below is a protocol using reversed-phase MicroPure column (MP-1602) for the cleanup of 5' Thiol oligonucleotides. Each MicroPure column will clean up to a 1 µmol synthesis column. Note that the TFA step is omitted and a 100 mM DDT in 0.2 M phosphate buffer pH 8.5 solution is substituted. The DTT solution will cleave the disulfide linkage resulting in a 5' Thiol modification.
1. Rinse column with MeCN. (2 x 2 mL)
2. Activate Column with 1 N TEAA. (3 x 2 mL)
3. Add Crude DMT-Oligo, 1 mL of 1:1 solution (conc. aq. 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. Cleave disulfide linkage with 100 mM DTT, in 0.2 M phosphate buffer (pH 8.5) (1 x 1 mL) Wait 30 minutes.
7. Rinse excess DTT with 5% MeCN in 0.1M TEAA. (3 x 2 mL) Rinse until pH is neutral.
8. Elute and collect purified oligo with 20 % MeCN in water. (1 x 1.5 mL)
9. Speed vac sample
Preparation of Reagents
Five different solutions for the purification process need to be prepared. To store these solutions use clean glass bottles with plastic lined caps, 250 mL in capacity is a convenient size.
1. Acetonitrile (VWR, cat. number JT9018-3).
2. 1.0 N Triethylammonium acetate (TEAA). For this solution, place about 80 mL of the water from #1 above in a clean bottle, and add exactly 5.7 mL of acetic acid (Aldrich cat. number 24, 285-3; Caution - causes burns, wear eye protection and gloves when using concentrated acetic acid). Swirl the solution until completely mixed, and add exactly 13.9 mL of triethylamine (Aldrich cat. number 23, 962-3; Caution - wear eye protection and gloves when using concentrated triethylamine). 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).
3. 3.0% Ammonium Hydroxide in water. Add 10 mL of concentrated ammonia (Aldrich cat. number 22, 122-8 Caution - wear eye protection and gloves when using concentrated ammonia) to 90 mL of water. Mix the solution until it is completely homogeneous.
4. 100 mM DTT solution in 0.2 M phosphate buffer pH 8.5
5. 5% MeCN in 0.1 M TEAA Use 5 mL MeCN in 95 mL 0.1 M TEAA.
6. 20% acetonitrile in water. Dilute 20 mL of the same acetonitrile as in 2 above with 80 mL of water. Image of cleaved and deprotected structure: The mass this product adds after conjugation and work-up (the additional mass seen by mass spectrometry) is: 328.43 |
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