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Novex 6 dna retardation gel

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The Novex 6% DNA Retardation Gel is a pre-cast polyacrylamide gel designed for the analysis and separation of DNA fragments. It provides a consistent and reliable platform for the electrophoretic separation of DNA samples.

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Lab products found in correlation

Sybr green 1 nucleic acid gel stain, by Thermo Fisher Scientific (2 mentions) Sds page gel, by Bio-Rad (1 mentions)

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6% DNA retardation gel (70 citations) Novex gels (17 citations) 6% retardation gel (3 citations) 6% DNA Retardation (3 citations)

3 protocols using novex 6 dna retardation gel

1

DNA-Protein Binding Assay for Apoptosis and ARM81 Regulation

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DNA probes PR63, PR72, PR-Apop and PR-ARM81ld were generated using plasmids JSS-3131, JSS-3129, JSS-3346k and JSS-3348k, respectively, and the primers listed in Supplementary Table 5. Each EMSA reaction contained 20 ng of DNA probe (8–14 nM depending on the probe). The concentration of cIApop and cIARM81ld protein used in each reaction ranged from 200 nM to 800 nM. Higher concentrations of the cI63 and cI72–HALO–HIS proteins (800 nM to 3,200 nM) were required owing to their limited solubility. The cI proteins with their respective probes were combined in binding buffer (50 mM NaCl, 20 mM Tris pH 7.5, 1 mM TCEP) and incubated at room temperature for 15 min. The samples were subjected to electrophoresis on a Novex 6% DNA retardation gel (Thermo) in 1× TBE at 100 V for 45 min. Double-stranded DNA was stained with SYBR Green I nucleic acid gel stain (Thermo) for 20 min. After washing with 1× TBE, gels were imaged using an ImageQuant LAS 4000 imager under the SYBR Green setting.
Silpe J.E., Duddy O.P., Johnson G.E., Beggs G.A., Hussain F.A., Forsberg K.J, & Bassler B.L. (2023). Small protein modules dictate prophage fates during polylysogeny. Nature, 620(7974), 625-633.
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2

EMSA Assay for cI-HALO Binding

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Primers JSO-956/957 (Table S2) and purified VP882 DNA were used to make the probe (257 bp). 20 ng of probe was used in each EMSA reaction. The highest concentration of cIVP882-HALO, designated 25x (Figure 1B), was ~300 nM. The concentration of the HIS-Qtip-cIVP882-HALO complex was matched to that of free cIVP882-HALO (no HIS-Qtip) based on the fluorescence from the cIVP882-HALO-conjuaged Alexa660 dye present in both samples. A 5-fold serial dilution of protein was applied to indicated lanes. The protein and probe were combined in binding buffer (25 mM Tris-HCl pH 8, 50 mM NaCl, 1 mM DTT), and incubated at RT for 15 min. The samples were subsequently loaded onto a Novex 6% DNA retardation gel (Thermo) at 4°C and electrophoresed in 1x TBE at 130 V for 2 h. The gel was stained for DNA by SYBR Green I Nucleic Acid Gel Stain (Thermo) and washed in 1x TBE for 20 min. The gel was subsequently imaged using an ImageQuant LAS 4000 imager under the SYBR Green setting. A duplicate batch of the samples was also subjected to electrophoresis in a 4–20% SDS-PAGE gel (Bio-Rad) to verify the correct loading of the HALO-Alexa660-labeled cIVP882-HALO in the EMSA. The gel was imaged using an ImageQuant LAS 4000 and detection of HALO and SNAP as outlined below.
Silpe J.E., Bridges A.A., Huang X., Coronado D.R., Duddy O.P, & Bassler B.L. (2020). Separating Functions of the Phage-Encoded Quorum-Sensing-Activated Antirepressor Qtip. Cell host & microbe, 27(4), 629-641.e4.
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3

Promoter Region Amplification and EMSA

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The DNA corresponding to the promoter region of vqmR, ∼100 bp, was amplified using V. cholerae genomic DNA as a template. Where mentioned, protein was pretreated in binding buffer with 10-fold molar excess DTT or diamide in order to reduce or oxidize the protein, respectively. To initiate electromobility gel shift assays (EMSAs), 0.2 to 3.5 μM protein was combined with 30 ng probe DNA in binding buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl). Reactions were allowed to proceed at room temperature (RT) for 15 min. Samples were separated on a Novex 6% DNA retardation gel (Thermo) by electrophoresis in 1× Tris-buffered EDTA (TBE) at 100 V. Gels were subsequently incubated with Sybr green reagent, diluted in 1× TBE at RT for 25 min, washed with five successive rounds of ddH2O, and imaged using an ImageQuant LAS 4000 imager and the Sybr green channel setting.
Mashruwala A.A, & Bassler B.L. (2020). The Vibrio cholerae Quorum-Sensing Protein VqmA Integrates Cell Density, Environmental, and Host-Derived Cues into the Control of Virulence. mBio, 11(4), e01572-20.
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