We use quite a different protocol for OPC purification, the yield is 25-40%.
Notably, we do not use methanol, we never use TeaAc after we load the oligo
onto the column, we never wash with 11% acetonitrile after we load the oligos,
20% acetonitrile is used only at the very end to elute the purified product.
If your yield is lower than ours, let me know and I will e-mail you our
protocol. In any case, two things are extremely important (in my view):
1) Use 70 deg Celsius / 90 min deprotection and purify the oligos right
afterwards, DMT group is lost spontaneously during prolonged storage (such as
overnight or over the weekend) in NH4OH.
2) After loading the oligo onto the cartridge (column), collect the
flowthrough and load again. Repeat this 4 times, for the total of 5 loadings,
before washing.
Best regards,
victor, Alpha DNA
> ----- Original Message -----
> From: Mark Lively <mlively@wfubmc.edu>
> To: Recipients of ABRF List <abrf@aecom.yu.edu>
> Sent: 23 juin, 1999 15:43
> Subject: DNASYN: RP purification method
>
> > Hi,
> >
> > I would like to get some suggestions/feedback in regards to our
> > purification method for
> > "long" oligos (over 25bp). We have been having difficulty with low
> > yield using a reverse
> > phase cartridge (Waters Sep Pak C18). We synthesize the oligos leaving
> > the last DMT "on" and our protocol is as follows:
> >
> > After cleavage and deprotection with concentrated NH4:
> >
> > 1. Rinse column with 100% Methanol
> > 2. Rinse column with 0.1M Tea-Ac (Triethylamine / Acetic Acid pH 7.0)
> >
> > 3. Load Oligo onto column with 0.1M Tea-Ac
> > 4. "Remove failures" with 11% Acetonitrile in 0.1M Tea-Ac
> > 5. Remove DMTs with 1.5% TFA
> > 6. Rinse with 0.1M Tea-Ac to neutralize TFA
> > 7. Rinse with dH20 to desalt
> > 8. Elute from column with ~ 1ml 40% Methanol, then lyophilized to
> > dryness.
> >
> > Any suggestions or modifications to improve this protocol would be
> > greatly appreciated.
> >
> > Thank you.
> > Mark
> > --
> > Mark O. Lively, Ph.D.
> > Professor of Biochemistry
> > Wake Forest University School of Medicine
> > Medical Center Blvd.
> > Winston-Salem, North Carolina 27157
> > Voice: 336-716-2969
> > Fax: 336-716-7200
> > email: mlively@wfubmc.edu
> >