Irdye 700

Manufactured by Integrated DNA Technologies

The IRDye 700 is a near-infrared fluorescent dye developed by Integrated DNA Technologies. It is designed for use in a variety of biological applications that require fluorescence detection in the near-infrared region of the spectrum.

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Lab products found in correlation

Tbe gel, by Thermo Fisher Scientific (2 mentions) Odyssey emsa kit, by LI COR (1 mentions) Odyssey dlx infrared imaging system, by LI COR (1 mentions)

Close spelling variants of this product

IRDye 700 labeled primers IRDye® 700 fluorophore 5′-IRDye 700

3 protocols using irdye 700

Preparation of DNA Oligonucleotide Duplexes

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DNA oligonucleotides
(unmodified, 5′-end-labeled
with IRDye 700, and 5′-end-labeled with 6-carboxyfluorescein)
were obtained from Integrated DNA Technologies (Coralville, IA). Preparation
of the duplex substrates used in this study is described in the Supporting Information. The molecular weights
and extinction coefficients of the DNA substrates were calculated
on the basis of their sequences and the molecular weights and extinction
coefficients of any appended dyes.^{23 (link)} The
concentration of the oligonucleotide strands and DNA duplexes was
determined spectrally using their calculated extinction coefficients.

Sanyal S.J., Yang T.C, & Catalano C.E. (2014). Integration Host Factor Assembly at the Cohesive End Site of the Bacteriophage Lambda Genome: Implications for Viral DNA Packaging and Bacterial Gene Regulation. Biochemistry, 53(48), 7459-7470.

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Characterization of ZBTB1 DNA Binding

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IRDye 700 and competitor oligonucleotides were purchased from Integrated
DNA Technologies (IDT). Oligonucleotide sequences are provided in Table S5. EMSA assays
were performed according to Odyssey instructions. Briefly, oligonucleotides were
diluted in TE to a final concentration of 20 pmol/uL, and 100 pmol of F and R
primers were annealed by heating to 100C and left to slowly cool back to room
temperature. Annealed oligos were diluted 1:200 for a working stock solution.
Binding reactions contained 10X binding buffer, poly dI-dC, 25 mM DTT, 2.5%
Tween 20, relevant oligonucleotides and purified ZBTB1 zinc-finger domain
protein. Reactions were performed at room temperature for 30 minutes prior to
electrophoresis through a 6% TBE gel (Novex). Gels were imaged using the Odyssey
CLx.

Williams R.T., Guarecuco R., Gates L.A., Barrows D., Passarelli M.C., Carey B., Baudrier L., Jeewajee S., La K., Prizer B., Malik S., Garcia-Bermudez J., Zhu X.G., Cantor J., Molina H., Carroll T., Roeder R.G., Abdel-Wahab O., Allis C.D, & Birsoy K. (2020). ZBTB1 Regulates Asparagine Synthesis and Leukemia Cell Response to L-Asparaginase. Cell metabolism, 31(4), 852-861.e6.

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Enhancer Variant Binding Assay Using EMSA

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Nuclear lysates were prepared from TE-7 and Jurkat cells using our published protocol.³⁹ (link) EMSA was performed using the Odyssey EMSA Kit from Li-Cor. Double-stranded oligonucleotides with 5′ IRDye 700 (Integrated DNA Technologies) were designed containing allelic enhancer variants in their 35-bp hg19 sequence context, with the allele of interest centered in the oligo (sequences are provided in Data S2). EMSA reactions containing 1× Odyssey EMSA binding buffer, 2.5 mM DTT, 0.25% Tween 20, 1 μg poly D(I-C), 0.05% NP-40, 8 μg of TE-7 or Jurkat nuclear lysate protein, and 50 fmol of IRDye 700-labeled double-stranded oligo were incubated 20 min at room temperature, then separated on a 6% TBE gel (Invitrogen). Results were imaged using a Licor Odyssey DLx Infrared Imaging System.
Assays for assessment of rs7203793 binding by USF1 were performed as above, with the inclusion of 25 ng recombinant USF1-Myc-DDK (Origene TP319106) or 1 μg USF1 G-2 antibody (Santa Cruz sc-390027 X) in the EMSA reactions.

Shook M.S., Lu X., Chen X., Parameswaran S., Edsall L., Trimarchi M.P., Ernst K., Granitto M., Forney C., Donmez O.A., Diouf A.A., VonHandorf A., Rothenberg M.E., Weirauch M.T, & Kottyan L.C. (2024). Systematic identification of genotype-dependent enhancer variants in eosinophilic esophagitis. American Journal of Human Genetics, 111(2), 280-294.

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