Applications

CRISPR-Cas 9: Reagents for CRISPR-Cas9

Reagents and kits used in CRISPR/Cas 9

dA-PACE Phosphoramidite

Glen Research

Catalogue No.	Description	Pack Size	Price	Qty	Change to: £
10-1140-02	dA-PACE Phosphoramidite	0.25g	€120.00	Quantity	Add to Order

Description

dA-PACE Phosphoramidite

Structure

Catalog Number: 10-1140-xx

Description: dA-PACE Phosphoramidite

5'-Dimethoxytrityl-N-benzoyl-2'-deoxyAdenosine, 3'-O-(N,N-diisopropylamino)-phosphinyl-1,1-dimethyl-2-cyanoethyl acetate
Formula: C₅₁H₅₈N₇O₈P	M.W.: 928.02	F.W.: 354.24

Diluent: Anhydrous Acetonitrile

Coupling: 33 minutes with 1H-Tetrazole Activator.
Synthesis: Oxidize before capping. Use either low water, low iodine oxidizer (Cat.# 40-4032-xx) or 10-camphorsulfonyloxaziridine (CSO) in acetonitrile. When CSO is used, extend the oxidation time to 3 minutes. Use 0.5M CSO when oxidizing mixed PACE/phosphodiester linkages. When oxidizing only PACE linkages, 0.1 M CSO can be used. For Cap Mix B, use 6.5% DMAP in THF (Cat.# 40-4020-xx). An example of a PACE cycle for AB synthesizers can be seen at:http://www.glenresearch.com/Technical/PACE_cycle.pdf

Deprotection: Pretreat synthesis column with 1.5% DBU in anhydrous acetonitrile for 60 minutes (syringe method). Wash with acetonitrile and dry with argon. Deprotect with 40% Methylamine for 20 minutes at 55°C.

Storage: Freezer storage, -10 to -30°C, dry

Stability in Solution: 2-3 days

Please Note: This product is covered by US Patents 6,693,187 and 7,067,641, patents pending and foreign counterparts owned by Lievre Cornu. Purchase of all or any of these products includes a limited license to use the product solely in the manufacture of oligonucleotides for RESEARCH USE ONLY and its use is not authorized nor intended for diagnostic or therapeutic use.

Please see Terms and Conditions of Use

PACE phosphoramidites

Phosphonoacetate (PACE) modified oligonucleotides show great potential as biological modifiers in a wide variety of research applications. PACE monomers are part of a family of Phosphonocarboxylate monomers. The monomers can be easily incorporated into complex oligonucleotides and are compatible with a wide variety of other sugar or heterobase modifications. PACE DNA can be conjugated through the carboxylic acid functional group. They have been shown to be active in siRNA duplexes and accelerate the initial rate of cleavage by RNase H-1 when incorporated with phosphorothioates. However, the most interesting observation to date is that they exhibit an unprecedented enhancement in penetration of cultured cells.

The phosphonoacetates are fully soluble in acetonitrile at a recommended concentration of 0.1M and are compatible with standard DNA synthesizers. A recommended coupling time of 33.3 minutes with 1H-Tetrazole is necessary when using the standard protocol. A modified LV cycle for AB instruments that reduces coupling time to 15 minutes with 1H-Tetrazole is available on our website. Oxidation must precede capping in the synthesis cycle. Reagents for oxidation depend on the type of synthesis. For fully modified oligos, we recommend the non-aqueous oxidizer camphorsulfonyloxaziridine (CSO) as a 0.1M solution. For mixed phosphodiester and phosphonoacetate modified oligos, a 0.5M CSO solution with a minimum oxidation time of 3 minutes is recommended. Low water oxidizer, 40-4032, is an alternative oxidizing reagent although it has been reported that this can result in conversion of a small percentage of the phosphonoacetate to the phosphodiester. We also recommend the use of the Cap Mix B with DMAP (40-4020) instead of the standard Cap Mix B containing 1-Methylimidazole.

The standard protocol for cleavage and deprotection requires a two step method with pretreatment using 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU) and subsequent cleavage using methylamine. The DBU is used to deprotect the dimethylcyanoethyl (DMCE) protecting groups and to prevent alkylation of the bases during deprotection. Cleavage with 40% methylamine in water is recommended and we have also had good results when using AMA deprotection.