Ttle. Use the same displacement technique in reverse, pushing the ACN into the phosphoramidite vial while it is still crimped and releasing the plunger with the needle in the headspace to allow argon to backfill the syringe. After repeating the push-release displacement a few times, the ACN will be transferred to the monomer vial without a significant build up of pressure.
2.
It is of little surprise, then, that during the humid summer months low coupling efficiency can be problematical. As such, steps must be taken to reduce the water content of all the reagents involved in the coupling step the acetonitrile (ACN) on the synthesizer, the ACN used to dilute the phosphoramidites, the activator solution (1H-tetrazole, DCI, ETT or others), and even the argon or helium used on the synthesizer, which should be dried with an in-line drying filter before reaching the synthesizer. And of course, the phosphoramidites themselves should be dry. 14
at capping than others. Our in-house work indicates that an ABI 394 caps total failures (generated by injecting ACN instead of monomer solution) with about a 97% efficiency, while an Expedite 8909 only caps at 90% efficiency in these tests. Part of this disparity is due to the concentration of N-methylimidazole in their respective Cap B mixes. For an ABI, it is normally a16% solution, whereas it is 10% for an Expedite. Indeed, we found that when 10% N-methylimidazole mix is used on the ABI 394, the capping efficiency drops to 89%. As a result, we recommend increasing the delivery of the Cap A/B mix on Expedites by 50% (going from 8 to 12 pulses) as well as increasing the time interval by 50% (going from 15 to 22 seconds). The most efficient capping reagent though is 6.5% DMAP solution used for Cap B. When this is used on an ABI 394, the capping efficiency jumps to 99%. While early work suggested that capping with DMAP could lead to a fluorescent adduct (ABI Nucleic Acid Research News, 7, Oct 20, 1988), we have never observed this side-reaction. suPPorts The supports themselves are critical to maintaining a high coupling efficiency throughout the sequence.937272-79-2 custom synthesis It is for that reason that we report the recommended synthesis length of every batch of support.
2.
This number reflects the drop-off point of the CPG and indicates when the pores of the suport are beginning to become essentially `clogged’ with nascent DNA strands. This leads to a drop in the coupling efficiency because the reagents are unable to diffuse quickly enough to pass through the nascent DNA strands before the next step in the synthesis cycle.133407-82-6 Synonym For very long oligos (100), we generally recommend a 2000 support.PMID:20301787 The difficulty of using 2000 CPGs is that they are quite friable, which can lead to 3′ base deletions, and have very low loadings (10 – 20 oles/g), which means it is generally not possible to synthesize oligos at a 1 umole scale. However, polystyrene (PS) supports are generally also good for long oligonucleotide synthesis and can be a worthy alternative. Indeed, it has been argued that it is easier to make the hydrophobic PS anhydrous prior to the coupling step. side reactions Depurination Even if the coupling efficiency is high and maintained throughout the length of the sequence, there are a number of side reactions that can lead to poor quality oligos. The most prominent of these is depurination. Trichloroacetic acid (TCA), which is the standard acid used in deblock solutions, is quite strong, with a pKa of approximately 0.7. Detritylation.MedChemExpress (MCE) offers a wide range of high-quality research chemicals and biochemicals (novel life-science reagents, reference compounds and natural compounds) for scientific use. We have professionally experienced and friendly staff to meet your needs. We are a competent and trustworthy partner for your research and scientific projects.Related websites: https://www.medchemexpress.com