Th two very hydrophobic dyes such as Cy5 and Dabcyl can require up to 25% ACN in 0.1 M TEAA. Finally, if the oligo does not bind quantitatively to the cartridge during loading, lower the pH by adding a few drops of glacial acetic acid. Conversely, if there is difficulty getting all the purified oligo off the cartridge when eluting, add a drop of 30% ammonia to the 50% ACN.
FRET Probes
Solution A: ACN : 2% TFA (1:2) Solution B: 12% ACN in 0.1 M TEAA
3′-Dye-Labelled Oligos
Solution A: ACN : 2% TFA (1:3) Solution B: 10% ACN in 0.1 M TEAA (If significant TAMRA or fluorescein is seen coming off cartridge, stop the rinse.)
overloading the cartridge results in lower probe yield and purity. Other dye combinations may require more stringent wash conditions depending upon the hydrophobicity of the dyes. Tetramethylrhodamine (TAMRA) dyes are sensitive to ammonia and will degrade in its presence; use UltraMILD DNA monomers (see `UltraMild DNA Synthesis’ in the Glen Research catalog) and deprotect in 50 mM K2CO3 in anhydrous methanol for 4 hours at room temperature. Using 0.02 M I2 for phosphite oxidation will lead to a cleaner probe. 3′-Dye-labelled Oligonucleotides This protocol has been optimized for 20mers labeled with either TAMRA or fluorescein on the 3′ terminus. The loading used was 50-60 OD per Poly-Pak cartridge (approximately 0.5 ole oligonucleotide). For longer oligos, or oligos that are labelled with a more hydrophobic dye, e.g., Dabcyl, use a higher ratio of ACN to 2% TFA in Solution A.
MERMADE INSTRUMENTS
The MerMade DNA synthesizer is a 192 well parallel array synthesizer developed at the McDermott Center for Human Growth and Development,an organized research unit of the University of Texas Southwestern Medical Center at Dallas. Assembled instruments may be purchased from BioAutomation in Plano, TX. Their web site can be found at: http://BioAutomation Phosphoramidite monomers are packaged in 240mL amber bottles for dissolving at the rate of 1g/20mL and are connected directly to the instrument. All solvents and reagents are prepared to our exacting specifications to ensure the highest synthesis efficiency and are passed through a 0.2 micron filter during packaging to eliminate particulate contamination. Tetrazole in activator solutions is typically at a concentration of around 0.475M, which is close to saturation in acetonitrile. In parallel synthesizers, there is a good possibility that tetrazole will crystallize on tips. This situation would lead to termination of flow which is bad in itself but, even worse, the blockage may be temporary.1201438-56-3 Formula A solution to this problem is to use 4,5dicyanoimidazole (DCI) as activator.36791-04-5 SMILES DCI is typically used at a concentration of 0.PMID:20301707 25M in acetonitrile which is far below its saturation level at greater than 1.1M. Regular 500supports may be used to fill the wells. However, this requires each plate to be prepared with each nucleoside accurately in all wells. A universal support clearly removes the need for four specific supports and makes preparing plates straightforward. Our universal support has been used in this type of synthesizer. The conditions to eliminate the terminal phosphodiester linkage entirely to 3′-OH are very forcing (e.g., ammonium hydroxide at 80 for 8 hours minimum). However, the use of ammonium hydroxide for 6 hours at 55 gives around 50% 3′-OH and oligos prepared this way are perfectly usable sequencing primers. For more stringent applications, the use of 0.4M sodium hydroxide in aqueous metha.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
