Irdye 700 labeled primers

Manufactured by Integrated DNA Technologies

IRDye 700 labeled primers are fluorescent DNA oligonucleotides designed for use in various molecular biology applications. These primers are labeled with the near-infrared dye IRDye 700, which enables sensitive detection and visualization of DNA amplification or hybridization products.

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Lab products found in correlation

Odyssey clx imaging system, by LI COR (2 mentions) Qiaquick pcr purification kit, by Qiagen (2 mentions)

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IRDye 700 Labeled primers

2 protocols using irdye 700 labeled primers

Electrophoretic Mobility Shift Assay for DNA Binding

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To assess DNA binding of Rv0500A, promoter regions for rv2390c (full length—704 bp; 5’ section—403 bp; 3’ section—301 bp) and kdpF (754 bp) were amplified using IRDye 700 labeled primers (Integrated DNA Technologies) and the PCR products purified using a QIAquick PCR purification kit (Qiagen). Indicated amounts of purified His-Rv0500A were mixed with no more than 40 fmoles of DNA in EMSA buffer (20 mM Tris-HCl, pH 8, 50 mM KCl, 2 mM MgCl₂, 5% glycerol, 0.5 mM EDTA, 1 mM DTT, 0.05% Nonidet P-40, 25 μg/ml salmon sperm DNA [65 (link)]) in 11 μl final volume reactions. After incubation at room temperature for 20 minutes, the reactions were run on a non-denaturing 8% Tris-glycine gel in HEPES-imidazole buffer (35 mM HEPES, 43 mM imidazole). The gel was then imaged using the 700 nm channel of an Odyssey CLx imaging system (LI-COR).

MacGilvary N.J., Kevorkian Y.L, & Tan S. (2019). Potassium response and homeostasis in Mycobacterium tuberculosis modulates environmental adaptation and is important for host colonization. PLoS Pathogens, 15(2), e1007591.

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Characterizing Transcriptional Regulators with EMSAs

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Electrophoretic mobility shift assays (EMSAs) were conducted as previously described (MacGilvary et al., 2019 (link)). Promoter regions of ftsK (771 bp), ftsW (717 bp), erp (746 bp), ripA (694 bp), and rv2390c (704 bp) were amplified using IRDye 700 labeled primers (Integrated DNA Technologies). PCR products were purified using a QIAquick PCR purification kit (Qiagen). Recombinantly purified Rv0500A (WT or M39A/T40A mutant) was mixed with no more than 30 fmol of DNA for 20 minutes at room temperature as previously described (MacGilvary et al., 2019 (link)). For competition EMSAs with the unlabeled probe, 200 fmol of unlabeled DNA was included in the mix with 1 fmol of labeled DNA and 0.1 μM of recombinantly purified WT Rv0500A. Samples were then loaded onto a non-denaturing Tris-glycine gel (5%) in HEPES-imidazole buffer (35 mM HEPES, 43 mM imidazole) and imaged after running using the 700 nm channel of a LI-COR Odyssey CLx imaging system.

Kevorkian Y.L., MacGilvary N.J., Giacalone D., Johnson C, & Tan S. (2022). Rv0500A is a transcription factor that links Mycobacterium tuberculosis environmental response with division and impacts host colonization. Molecular microbiology, 117(5), 1048-1062.

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