FREQUENTLY ASKED TECHNICAL QUESTION
QUESTION: How do the 1000Å and 2000Å supports compare in the synthesis of long oligos? Do you recommend any changes to the cycle for long oligos?
RESPONSE:In one comparison several years ago, a customer compared 1000Å, 2000Å and a low-loaded 500Å CPG for the synthesis of 200 and 400mers. Only in the case of the 2000Å CPG could the 200mer product be seen on a gel although the other supports did make product since it could be amplified by PCR. The 400mers could not be seen but could be amplified by PCR.
For the synthesis of long oligos, we recommend increasing the coupling wait step from 15 seconds to at least 30 seconds. I believe this is important later in the synthesis. Also, we recommend the use of DMAP in the Cap B solution or, if methylimidazole has to be used, increasing the capping wait step to 45 seconds. With incomplete capping, you are going to be making oligos contaminated with deletion mutations. I know DMAP has been accused of causing base modification of dG sites leading to fluorescent bands on gels but it is still the most effective capping activator. I also would recommend, if possible, an extended capping of the support (20 minutes) before the DMT is removed in the first cycle. The 1000Å and especially the 2000Å supports are very fragile and can be damaged in transit. This capping step deactivates any fresh surfaces caused by fractures. TWIST columns are assembled with 20µM frits to retain any fines coming from these friable supports.
J.S. Eadie and D.S. Davidson, Nucleic Acids Res., 1987, 15, 8333.
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