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| The scale of synthesis refers the amount of the first "base" used at the start of the DNA synthesis. | |
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There are several steps to DNA synthesis, including: |
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| Since none of these steps is 100% efficient, the actual yield of oligonucleotide will be less than the scale at which it was synthesized. Oligonucleotide synthesis starts at the 3'-terminal base, which is attached to the solid support. Bases are added one at a time in the 3'-end to 5'-end direction. For each coupling/capping reaction, the efficiency of the reaction is between 97.0 - 99.5% efficient; we observe an average coupling percentage of 98.5%. | |
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For example, at the end of the synthesis of a 20-mer, which has undergone 19 coupling reactions, the amount of full-length material ranges between 0.9720 to 0.9920, or 54% to 91% (average = 74%). The remaining 9 - 46% of the material would consist of approximately equal amounts of the 19-mer, 18-mer, 17-mer, etc. |
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Following synthesis, the oligonucleotide is deprotected in concentrated ammonium hydroxide and desalted on a gel filtration column. If desired by the customer, additional HPLC or PAGE purification can also be performed. The desalting and additional purification steps are not 100% efficient. Depending on the length of the oligonucleotide, the complexity of the synthesis, and the type of purification, the yield from a 0.2 micromole scale synthesis can be as low as 10 nanomoles or greater than 100 nanomoles. |
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