A branching monomer is required to construct comb-like oligonucleotide probes. The developers of the comb system from Chiron Corporation evaluated1 several protecting groups for the branch point and chose levulinyl (LEV), which is specifically removed using a reagent containing hydrazine hydrate, acetic acid and pyridine.
Details
Usage
Coupling: dC Brancher reacts in a manner identical to normal phosphoramidites.
Deprotection: Levulinyl Deprotection: The levulinyl protecting group can be selectively removed without cleavage of the oligonucleotide from the CPG by treatment with 0.5 M Hydrazine hydrate in 1:1 pyridine/acetic acid. [Note hydrazine hydrate is a violent poison that is both volatile and readily absorbed through skin] Fit the column with syringes and push the solution back and forth across the column. Let sit for 15 minutes at room temperature. Rinse the column with 1.5 mL of 1:1 pyridine/acetic acid (3x) and then 1.5 mL of ACN (3x). Dry CPG under a stream of argon and proceed with the synthesis of the branching sequence. If a non-branching control is desired, simply deprotect in ammonium hydroxide as required by the nucleobases.
Specifications
Diluent
Anhydrous Acetonitrile
Storage
Refrigerated storage, maximum of 2-8°C, dry
Stability
24 hours
Dilution/Coupling Data
The table below show pack size data and, for solutions, dilution and approximate coupling based on normal priming procedures.
ABI 392/394
Catalog #
Pack Size
Grams/Pack
0.1M Dil. (mL)
Approximate Number of Additions
LV40
LV200
40nm
0.2μm
1μm
10μm
10-1018-02
0.25 g
.25grams
2.65
75
45
28.13
20.45
15
3.75
10-1018-90
100 µmol
.094grams
1
20
12
7.5
5.45
4
1
Expedite
Catalog #
Pack Size
Grams/Pack
Dilution (mL)
Approximate Number of Additions
Molarity
50nm
0.2μm
1μm
15μm
10-1018-02
0.25 g
.25grams
3.96
0.07
72.8
45.5
33.09
4.55
10-1018-90
100 µmol
.094grams
1.5
0.07
23.6
14.75
10.73
1.48
References
REFERENCE(S)| 1 T. Horn, C.A. Chang, and M.S. Urdea, Nucleic Acids Res, 1997, 25, 4842-4849.
