1) OPC or equivalent techniques are NOT APPROPRIATE for such long
oligos.
2) IEX could work but I would recommend 2 successive injection. We used
this
with some success (Protein-Pak Q8HR 8=B5m 10x100 mm from Waters)
NaCl gradient (100 mM to 1M within 50 minutes) in 10 mM NaOH (helps to
denaturate oligo structure).=20
3) If small amount of oligo are necessary, I would recommend PAGE
purification
on 10% acrylamide gel and in denaturing conditions (7M urea in the gel
and=20
75% formamide in the loading buffer - Boil the oligo for 5 minutes and
chill in
ice immediately after boiling). Quite typical DNASEQ gel conditions.
The oligos are revealed by UV shadowing and=20
the piece of gel is incubated at 37 C overnight for extraction. Yhe
oligo
is then desalted on G25. According to our experience, it's the best.
4) Comment : What means >95% pure ? It depends on the analysis system.
Very easy to show >95% on RP (C18) HPLC, could be much different if
analysis is done on IEX with isocratic conditions or on PAGE after=20
radiolabeling of the oligo. Which 95% is necessary is an important
question=20
also !
And I will take the risk to look arrogant but may I remind that the
better
your synthesizer works, the better you purified oligo will be. When we
have to produce very pure long oligos the synthesizer is checked,
calibrated
extensively and only fresh products from best source are used. But I
guess
everybody knows that. Sorry If I hurt with this comment.
I hope it will be helpfull.
--=20
Marc LEMAITRE, Ph.D.
EUROGENTEC Bel S.A.
Parc de Recherche Cense Rouge
B-4102 SERAING (BELGIUM)
Tel : (+ 32) 4 366 01 50=20
Fax : (+ 32) 4 367 45 00
E-mail : <mailto:m.lemaitre@eurogentec.be>
Web site : <http://www.eurogentec.be> <-------- check it out !