Dear all,
Currently I am using DNA synthesizer (ÄKTA oligopilot plus) to synthesize oligonucleotide(20-50nt), whether phosphodiester and phosphothiolated. However, I've been stucked in the purification process for a while. Our lab use HPLC for separation and purification. We have two C18 column for this, semi-preparative and preparative. I used the semi one more often. The flow phase I set is acetonitrile and water(0.1M TEAA), 95%-70%, based on the experience from other lab in our college. After preparation, the oligos were detached from CPG and went HPLC for separation of the product from failure strand. Then the product was lyophilized, deprotected and purified by HPLC. The problem lies in the separation process: it is quite hard to determined the peak of my product, specially for phosphothiolated oligonucleotide. I'm wondering if it is the problem of my HPLC method, or the C18 column is not suitable for the separation and purification of oligonucleotide (specially larger than 40nt). BTW, is there any other method that could be applied?
Thanks,
Guangqi
Dear Guangqi Wu,
Look the link. May be useful.
http://www.sigmaaldrich.com/technical-documents/articles/biology/best-purification.html
https://www.idtdna.com/pages/products/dna-rna/hplc-page
Regards, Shafagat
Dear Guangqi,
Prerequisite for a HPLC purification is a DMT-ON synthesis. I think you have done this.
We have a long-time experience and the best results with this resin, originally developed by ABI:
https://www.thermofisher.com/order/catalog/product/1133903 or google with Oligo R3.
You can fill the columns easily by yourself under normal conditions. You need no high pressure for this and you can use the columns for ~1000 runs. You can use your gradient system. In an analytical run with a small amount of your oligo mixture you can find the best conditions for separation (time and gradiend increase). The material is suitable also for phosphorothioates and a lot of other 5' or 3' modifications. For your further questions you can contact me directly under:
I hope, I could help you
Best regards
Gottfried
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