Nucleic acids direct elution

P Bridonneau, YF Chang, AV Buvoli, D O Connell, D Parma. Site-directed selection of oligonucleotide antagonists by competitive elution. Antisense Nucleic Acid Drug Dev 9 1-11, 1999. 

Finally, the transcripts were completely digested by RNase Ti at 37°C for 10 min with MALDI matrix (3-HPA) added as a denaturant. Briefly, 5 pL of RNA transcript (up to 20 pg) is added to 4 pL 3-HPA in 50% ACN/water and 1 pL of RNase Tj (1000 units) and reacted for 10 min and then placed on ice for MALDI preparation. For highest quality spectra, samples are desalted by reverse-phase purification in ZipTips according to manufacturer s directions for nucleic acid purification [96]. The final step in this process is elution of purified RNA oligonucleotide fragments onto the MALDI target using 2 pL of the MALDI matrix itself. Samples (MALDI spots ) are allowed to air dry or may be rapidly dried under vacuum. 

Pataki, G., and Niederwieser, A. (1967). Thin-layer chromatography of nucleic acid bases, nucleosides, nucleotides and related compounds. IV. Separation on PEI-cellulose layers using gradient elution and direct fluorometry of spots. J. Chromatogr. 29 133-141. 

Mass spectrometry (MS) is used for nucleic acids, but typically after zonal elution to avoid interfering solutes. Present developments in ion coupled MS/TLC must take into account the sorbent, solvent, and analyte, which will not exceed 0.25% w/w based on sample and sorbent (8,64). The apparatus requires the ability to extract, elute, or volatilize analyte directly from the TLC plate. When available, these instruments will be helpful to detect and characterize analytes. 

Purification of the enzyme from extracts of E. coli was carried out by precipitating nucleic acids with MnCU, adsorbing the enzyme on calcium phosphate gel and eluting with phosphate buffer, and then fractionation with (NH4)sS04 between 0.32-0.45 saturation. This procedure produced roughly a tenfold enrichment of enzyme activity. The reductase has its pH optimum at about 8.5. Michaelis constants at pH 8.0 were determined to be 5.5 X 10 M for shikimic acid and 3.1 X 10 M for TPN. (Because of the scarcity of dehydroshikimic acid, the reaction was followed only in the reverse direction.)... 

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