oligo deprotection

Chia Jin Ngee mcblab47 at leonis.nus.sg
Fri Jun 3 22:50:19 EST 1994


Dr. A. Rosenthal (arosenth at crc.ac.uk) wrote:
: Hi everybody,
: somebody just told me that he had heard, one could deprotect oligos by boiling them
: for a short while!? rather than the 5hour or o/n 55'C waterbath.
: Any comments greately appreciated.
: Thanks.

Yes you can. But not boiling them. I use a Milligen 7500 DNA Synthesizer. 
This is the procedure I follow:

1. After systhesis, dry the CPG in the column under vacuum.

2. Remove the CPG from the column and put it in a 2-ml screw-cap 
microfuge tube. I use Sarstedt ones.

3. Fill it with conc. ammonia solution almost to the top and add 100 ul 
of TEA.

4. Incubate at 80 to 85 deg C for 30 min to 1 hour. Make sure the 
screw-cap is tight but not over tightened, otherwise the ammonia solution 
boils out taking your precious oligo with it. I usually use a heat block 
for this purpose. The timing depends. For an oligo less than 30-mer, 30 
min would be fine. Anything greater than 30-mer would require longer 
times. I have simplified this and deprotect <30-mers for 30 min and 
>30-mers for 1 hr. 50-mers and greater would require 1 hr 30 min.

5. Cool the tube after the deprotection in a freezer for 30 min.

There! Deprotected oligo in less than 2 hours.

Under HPLC analysis, the oligos are no different from normally deprotected
ones. However, shorter oligos <10-mers tend to be slightly depurinated.
This is not much of a problem

--
Jin Ngee, Chia			Chemical Carcinogenesis Laboratory
(Genie, the OligoMan)		Institute of Molecular and Cell Biology
mcblab47 at leonis.nus.sg		National University of Singapore
Tel: (065)772-3797		Kent Ridge Crescent
Fax: (065)779-1117		Singapore 0511
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"I felt the sword lifted from my hand when He (Christ) died."
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