This study elucidates the optimum conditions at the minimum cost for using SYPRO Ruby protein gel stain. It deals with the effects of gel fixation and staining times, as well as dilution and reuse of SYPRO Ruby protein gel stain in one-dimensional (1-D) gels. Signal strength and dynamic range were highest in gels that were fixed thoroughly before staining, followed by overnight staining. Using the optimized protocol, dilution or reuse of the stain reduces the dynamic range and signal intensity. Sensitivity remains high if the stain is reused up to two times, but signal intensity is reduced up to 2.5-fold in twice used stain. Sensitivity also remains high if the stain is diluted 1:2 in water, but signal intensity is reduced up to 6-fold. Of the two options, reuse or dilution, reuse better retains signal intensity and dynamic range.
We have identified a complex between TFIIIB and the upstream promoter of silkworm tRNA Ala genes that is detectable by gel retardation and DNase I footprinting. Formation of this complex depends on the integrity of previously identified upstream promoter elements and on the presence of other silkworm transcription factors, either TFIIID or a fraction that contains both TFIIIC and TFIIID. We have used this complex to compare the interactions of TFIIIB with two kinds of tRNA Ala genes whose different in vitro transcription properties are conferred by the upstream segments of their promoters. These are the tRNA C Ala genes, which are transcribed constitutively, and the tRNA SG Ala genes, which are transcribed only in the silk gland. We find that TFIIIB binds tRNA SG Ala genes with lower affinity than it binds tRNA C Ala genes. In addition, the TFIIIB complexes formed on tRNA SG Ala genes differ qualitatively from those formed on tRNA C Ala genes. Both the transcriptional activity of tRNA SG Ala complexes and the ability of the complexes to protect upstream DNA from DNase I digestion are reduced.
Quantification of DNA or RNA is often confounded by source contamination or inefficient purification. In addition, limited or sensitive samples may often force scientists to forgo quantification in order to avoid the potential of contamination. Here we describe the development and validation of three fluorescent reagents selective for the detection of DNA and RNA that are resistant to common sample contaminants such as salt, nucleotides, and protein. Moreover, the dsDNA reagents are resistant to contamination from different nucleic acid contaminants (such as nucleotides or RNA) while retaining their sub‐nanogram sensitivity. Likewise the RNA reagent is resistant to contamination from ssDNA, oligos and dsDNA while still able to detect low nanogram quantities of RNA. Compatible with either high‐throughput microplate based fluorometers, or low‐throughput single cell fluorometers, the assays were developed specifically for pairing with the dedicated quantification fluorometer, the Qubit™ fluorometer. The Qubit fluorometer is programmed with a unique curve fitting algorithm that reduces the standards from the eight normally used in plate readers down to two while retaining the same precision and accuracy.
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