Recombinant SOX17-Flag and Flag alone (pcDNA3 vector (Promega)) were expressed in 293T cells. Plasmids were transfected using Lipofectamine 2000 Transfection Reagent (Life Technologies, 11668019) 36hrs before cells were lysed in RIPA buffer containing protease inhibitors. Recombinant protein was immunoprecipitated from lysate overnight at 4°C with Anti-FLAG M2 magnetic beads (Sigma, M8823) and the recombinant protein eluted with excess FLAG peptide. 5–7ul of the first eluate was used in a binding reaction along with 0.3pMol of complementary annealed 3′Biotin-labeled oligonucleotides (Integrated DNA Technologies), 300-fold excess competitor probes, 0.02U Poly(dG-dC) (Sigma, P9389), and binding buffer (100mM HEPES pH 8.0, 50mM KCl, 500 μM DTT, 50 μM EDTA, 1mM MgCl2, and 5% glycerol by volume)69 (link). DNA-protein complexes were resolved on 7% native polyacrylamide gel, transferred to neutrally charged nylon membrane, incubated with Streptavidin-POD (Roche, 11089153001) and imaged by chemiluminescence. See Supplementary Table 4 for probe sequences.
3 biotin labeled oligonucleotides
Manufactured by Integrated DNA Technologies
Sourced in United States
3′Biotin-labeled oligonucleotides are synthetic DNA or RNA molecules with a biotin molecule attached to the 3′ end. Biotin is a small molecule that can be used to detect or capture the oligonucleotide in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Poly dg dc, by Merck Group (1 mentions)
Streptavidin pod, by Roche (1 mentions)
Anti flag m2 magnetic bead, by Merck Group (1 mentions)
Pcdna3 vector, by Promega (1 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Terminal transferase, by New England Biolabs (1 mentions)
Biotin dutp, by Roche (1 mentions)
Cocl2, by New England Biolabs (1 mentions)
Tdt buffer, by New England Biolabs (1 mentions)
2 protocols using 3 biotin labeled oligonucleotides
1
Recombinant SOX17 Protein Purification and DNA Binding Assay
2
Exome Capture and Circularization Protocol
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The list of the entire exons for the 30 genes was obtained from a consensus coding sequence database, CCDS (build 36.3), showing a total number of 701 exons covering 102 kb, according to hg18 (March, 2006 assembly).
Targeted restriction fragments were selected using Disperse software [11 (link)]. Templates for circularization of each chosen targeted fragment (selector probes) were designed using ProbeMaker software [12 (link)]. Each selector probe consisted of two sequences of 20-25 nucleotides complementary to the ends of its targeted restriction fragment.
The 3'-biotin-labeled oligonucleotides (Integrated DNA Technologies, Coralville, IA, USA) were prepared by incubating the oligonucleotides with 1× Tdt buffer (NEB), 1× CoCl2 (NEB), 0.1 mM dUTP-biotin (Roche Diagnostics, Mannheim, Germany), and 0.2 units/µL terminal transferase (NEB) in a final volume of 50 µL. The reaction was incubated at 37℃ for 1 h and followed by enzyme inactivation at 75℃ for 20 min.
Targeted restriction fragments were selected using Disperse software [11 (link)]. Templates for circularization of each chosen targeted fragment (selector probes) were designed using ProbeMaker software [12 (link)]. Each selector probe consisted of two sequences of 20-25 nucleotides complementary to the ends of its targeted restriction fragment.
The 3'-biotin-labeled oligonucleotides (Integrated DNA Technologies, Coralville, IA, USA) were prepared by incubating the oligonucleotides with 1× Tdt buffer (NEB), 1× CoCl2 (NEB), 0.1 mM dUTP-biotin (Roche Diagnostics, Mannheim, Germany), and 0.2 units/µL terminal transferase (NEB) in a final volume of 50 µL. The reaction was incubated at 37℃ for 1 h and followed by enzyme inactivation at 75℃ for 20 min.
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