The neat teA.3Hf used in the original methods, which tended to both remove Dmt and depurinate dA sites in chimeric oligos. these cocktails also function well with all three types of RNA monomers available in the Glen Research catalog. In addition, it must be noted that tBAf is not compatible with the Glen-Pak RNA purification process.
deprotect to completIon Base Deprotection In this article, we will focus on regular base deprotection using ammonium hydroxide/methylamine (AmA) at elevated temperature, which we have shown to be optimal for both tom-protected and tBDms-protected RNA, and ultramild deprotection using ammonium hydroxide/ ethanol (3:1) at room temperature for oligos
4
Butanol Precipitation to complete the 2′ deprotection for a Dmt-off RNA, fully re-dissolve the oligo in anhydrous Dmso. Remember to avoid glass and use a sterile or RNase free, polypropylene, o-ring capped tube for this reaction. If necessary, heat the oligo at 65 for about 5 minutes to get the oligo fully into solution. Add teA.3Hf, mix well and heat to 65 for 2.5 hours. cool the solution and desalt the oligo via butanol precipitation. DMT-on Purification the 2′ deprotection of Dmt-on RNA is slightly different due to the addition of teA to the cocktail, thus aiding in retention of the Dmt. the deprotected RNA is then quenched and immediately purified on a Glen-Pak RNA cartridge. fully re-dissolve the RNA in anhydrous Dmso and if necessary, heat the oligo at 65 for about 5 minutes to get the oligo fully into solution. Add teA to the Dmso/ RNA solution and mix gently. follow this with teA.3Hf, mix well and heat to 65 for 2.5 hours. Instead of quenching the cocktail by the addition of butanol, add 1.75ml of RNA Quenching Buffer (60-4120-XX) to the reaction. the sample is now ready for GlenPak RNA cartridge purification. Glen-Pak Purification Glen-Pak RNA purification cartridges can purify from 40 nmole to 1.0 ole scale syntheses in one load and are supplied in two formats; one for vacuum manifolds and another for use with a disposable syringe. Glen-Pak purified RNA oligos routinely show purities of between 90 and 95% and yields in the 50 to 80 oD range for a 1.0 micromole synthesis. once the 2′ deprotection solution is quenched, immediately load the 2ml solution on a properly prepared Glen-Pak RNA cartridge and follow the Glen-Pak RNA procedure. At the end of the procedure, the oligo is eluted in 1.0ml RNase free 1m ammonium bicarbonate/30% Acetonitrile and lyophilized to dryness. Ammonium bicarbonate is a volatile salt, but a second drying step from RNase free water may be required to remove excess bicarbonate.1135243-19-4 medchemexpress RNA Analysis Accurate analysis of Glen-Pak purified RNA by Ion exchange HPlc using more traditional buffer systems can be hampered
Figure 1: ion excHange Hplc anD eSi mS of SyntHeSizeD Sirna ACC UUG AGG UAU ACU GCG ATT
Column: Dionex DNAPac PA200, 250 X 4mm; Buffers: A- 10mM NaClO4, 25mM TRIS-HCl, 20% Acetonitrile, pH 7.849214-04-6 Molecular Weight 4; B- 600mM NaClO4, 25mM TRIS-HCl, 20% Acetonitrile, pH 7.PMID:29763107 4; Gradient: 0-10% Buffer B over 30 minutes; Flow 1mL/min.
Target Mass(Da), 6670.1; Observed Mass(Da), 6669.3; Mass error: -0.8 Da (-0.012%), %Purity (Estimate) 93.55%,
by formation of secondary structures. the use of a sodium perchlorate buffer system as well as heat should denature most oligoribonucleotides. figure 1 shows the Ion exchange HPlc analysis of a Glen-Pak purified, 21-mer siRNA. Another way to avoid secondary structure issues and obtain proper identity determination of olig.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