Agarose quality

The in vitro transcribed RNAs are phenol-chloroform extracted, ethanol-precipitated, and washed once with 70% ethanol. The RNA pellet is resuspended in 30 1 H20 and loaded to DyeEx 2.0 spin columns (Qiagen) to remove free nucleotides the RNA is then quantified by spectrophotometry. Approximately 20 to 30 pg of RNA is obtained from one reaction. For the initial preparation of transcripts, we would examine the quality and quantity of synthesized RNA by separating them on 1% denaturing agarose gels with total cell RNA preparation as a size maker (28S and 18S ribosomal RNAs... 

The structures of agarose, iota- and kappa-carrageenan have been determined by x-ray fiber diffraction (24-27). The quality of the diffraction data obtained from each of these three specimens varies considerably and the way in which these data are used in structure determination is outlined here. Diffraction patterns from oriented specimens of agarose, and kappa- and iota-carrageenan are shown in Figure 6 (28). The molecular repeat distances derived from these patterns are listed in Table I. 

Resuspend the pellet in 6 pi of DEPC-treated ddH O. Use a 1-pl aliquot to determine the concentration with a spectrophotometer and check the quality of the RNA probe on an agarose gel see Note 9). 

Although the spectrophotometric measurements at different wavelengths can determine the extent of contaminants, the overall quality of DNA can be determined by analyzing samples on a horizontal 0.7% agarose gel... 

In cases where very small quantities of RNA are isolated, quality assessment can be made by probing Northern blots prepared with small quantities of RNA with probes such as ribosomal RNA, /i-actin, or oligo(dT). Such blots can be prepared by size fractionation of nanogram quantities of RNA in formaldeyde-agarose gels, followed by transfer to nylon membrane under high salt conditions [10,35],... 

Total RNA or (polyA+)RNA (mRNA) can be used for experiments. In the latter case, a purification step is necessary, as mRNA is isolated from total cellular RNA by affinity chromatography on oligo-dT immobilized to a solid support. The amount of the purified RNA is determined by its dual wavelength absorbance at 260 nm and 280 nm and the quality checked by agarose gel or capillary electrophoresis. 

Quantify total RNA using Eppendorf BioPhotometer and control quality by 1% agarose gel electrophoresis on Amilabo electrophoresis power supply ST 1006T. 

Collection and Treatment of Brain Samples Brains were obtained form animals, which were sacrificed 3 h post oral dosing of the drug. Brains of untreated (control) animals were used in order to obtain the blank brain homogenates for calibration standard and quality control samples (control animals received the pure vehicle, 0.2 % w/v agarose gel). 

Check the quality of the plasmids used, by electrophoresis on agarose gels. 

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