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Desalting Protocol
DESALTING PROTOCOL
Our unpurified product conatins various organic
salts, including benzamide, isobutyramide, and ammonium acetate which are utilized during
sythesis to protect both the backbone and the bases. The following procedure utilizes a
disposable reverse-phase cartridge to remove these salts.
ULTRAMILD OLIGOS: Before
desalting UltraMild oligos that have been cleaved and deprotected using
potassium carbonate in MeOH, the MeOH content must be reduced to < 5% through
speed-vac'ing or dilution with "A" Buffer. An organic content > 5% will prevent
DNA from binding to the Sep-Pak resin! Adding TE to UltraMild oligos is not
required.
Materials and Buffers:
- Sep-Pak Plus C18 Cartridge (Waters part #WAT20515)
- "A" Buffer (5mM triethylamine in
dH2O tritrated with glacial acetic acid to pH 7.0-7.5, or a 1:400 dilution in
dH2O of 2M Triethylamine acetate (ABI part#400613)
- "B" Buffer ("A" Buffer + 50% MeOH [final]=5mM
triethylamine/50% MeOH)
- 95% MeOH
- TE pH7.0
- stand with small clamp
- 10cc syringe
Procedure:
- Resuspend oligo in TE to a final concentration of 1-5 mg/mL
- Dilute an aliquot of the resuspended oligo 1:100 in TE; read
A260
- Secure Sep-Pak cartridge to stand with small clamp
- Wash cartridge with 7mL 95% MeOH using 10cc syringe (flow rate
= 1-2 drops/second for all steps)
- Wash column with 7 ml "A" Buffer
- Load a maximum of 50 O.D. units of resuspended oligo onto the
cartridge; collect eluant and monitor A260
- Wash bound oligo with 7mL "A" Buffer; collect eluant
and monitor A260
- Elute desalted oligo using 3mL"B" Buffer, collect
eluant and monitor A260
- Dry peak fractions in a Speed-Vac
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