In my experience, users who ask for 95% pure mean that they do not need it
that pure, not DNA repair study grade. Thus, if you like gels, do gels. If
you are set up for HPLC, do Trityl-ON plus Trityl-OFF reverse phase HPLC,
and your customer's applications will be fine. I would not recommend using
only one Trityl-ON step in case the synthesis is not as good as you
normally accomplish. A great purification does start with a great
synthesis. However, for a small amount of good DNA, if you do gels you do
not need a great synthesis, just a good one. The gel will resolve both n-x
and deprotection problems if the DNA band is run at least 20 cm into the gel.
If you do gels, use a 3 inch wide well in a 0.4 mm thick sequencing gel,
urea and 50 C and all that. Load about 10 A260 of DNA and run to 20 cm for
the main band. Cover the inside 80% of the band before you UV shadow to see
and mark the positions of the side fractions. I assume that you can run
gels without them smiling. Cut out the 80% that has not been exposed to UV.
Be brave and cut the UV dark band very thin. Your gel should have a
resolution as good as the sequencing gels you see in DNA sequencing. Elute
the DNA with electroelution in the Amicon unit into centricon 3, at 200V
for 2 hours. Concentrate and buffer exchange if necessary in the Centricon.
You will get over 95% recovery. The upper reservoir of the AMICON unit has
9 wells and can lead to cross-contamination if more than one oligo is
eluted at the same time. I have built my own reservoirs with isolated
reservoirs for each centricon, thereby avoiding contamination. Do treat the
upper reservoir with bleach to avoid contamination. Your DNA will be DNA
repair enzymology grade. I highly recommend the modified electroelution. If
you need specifics, email my associate Kate Oleykowski at
CA_Oleykowski@fccc.edu who is a hands-on expert at oligonucleotide gel
purification, having done numerous purifications of longmers for DNA repair
studies. You will be in good hands.
By HPLC or by gel,I am sure you will have a satisfied customer.
Tony
>On Mon, 2 Feb 1998, Mary Kay Dolejsi wrote:
>
>> What is the "best" method for purifying full-length long oligomers- like 80
>> - 85mers? A researcher here wants to make such a longmer, and have it >95%
>> pure. Can solid-phase methods, like OPC really do this? Can IEX resolve at
>> this length? Gel electrophoresis and elution?
>>
>> I appreciate any help you can give us. I know there was a recent
>> discussion about a 45mer, but has anyone been working with longmers?
>>
>> Thanks in advance.
>>
>>
>> Mary Kay Dolejsi, Ph.D.
>> marykay@fred.fhcrc.org
>> phone: 206-667-4470
>> fax: 206-667-6497
>>
>> Mail address:
>> Fred Hutchinson Cancer Research Center
>> 1100 Fairview Ave N. A1-162
>> PO Box 19024
>> Seattle, WA 98109-1024
>>
>>
>>
>
>
>
************************************
Dr. Anthony T. Yeung, Ph.D.
Director, Fannie E. Rippel Biotechnology Facility
Member, Institute for Cancer Research
Fox Chase Cancer Center
7701 Burholme Ave. Philadelphia, PA 19111
Voice: 215-728-2488
FAX: 215-728-3647
email: AT_Yeung@FCCC.edu
http://www.fccc.edu/research/labs/yeung/
************************************