I think that you may be losing some of you DMT on oligo in your 11% ACN/T=
EAAc wash step. I have used Sep Paks in the past and would have to perio=
dically have to vary the concentration in the ACN wash step. I don't kno=
w if this was due to changes in the packing material or due to concentrat=
ion changes in the 11% ACN/TEAAc solution. It is easy to evaluate the pe=
rformance of your procedure by analytical RP HPLC. =20
1. Analyze a crude oligo by RP HPLC and determine the % DMT-on content.
2. Load a known number of A260 units of the crude oligo on the column.
3. Collect the eluents from each step and analyze by RP HPLC and determin=
e if you are losing DMT-on oligo in the ACN wash step.
4. If you take into account the volumes of each wash step and dilutions o=
f the HPLC samples you can do a mass balance of DMT-on oligo and determin=
e the fate of the DMT-on oligo.
One other thing to look for. You may be losing DMT from your full-length=
oligo during the dry down step. Extended time in the Speed-Vac can resu=
lt in loss of trityl. I used to add 50 =B5l of TEA to each tube before d=
rying down. Otherwise don't dry the oligo completely to dryness. I hope=
this helps.
>Mark,
>
>One modification to that procedure is to remove the DMT oligo from the
>column/cartridge after you strip the failure products. Do the detritylat=
ion
>in a tube, not on the column/cartridge. Then precipitate the oligo and
>wash.
>
>Dick
>
>
>
>
>
>
>
>
>
>
>>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
>
Regards,
Bob
Robert L. Somers, Ph.D.
Sr. Applications Chemist
Glen Research Corporation
22825 Davis Drive
Sterling, VA 20164
+703-437-6191 (Voice)
+703-435-9774 (Fax)
http://www.glenres.com