The EMSA was carried out as previously described (Crane et al., 2018 (link)) except that the purified RecA protein was from New England Biolabs instead of Abcam (NEB cat. no. M0249). The 10 X reaction buffer supplied with the RecA was used. As previously described, the ssDNA probe was a fluorescent 39-mer oligonucleotide labeled with fluorescein (IDT DNA). The 100 bp DNA ladder was from Invitrogen/ThermoFisher, cat. no. 15628-019. Adenosine 5’-(gamma-thiotriphosphate), or ATP-γ-S, was from Sigma-Aldrich, cat. no. A1388. Fluorescent bands were imaged using the Gel Doc EZ instrument from Bio-Rad and using the corresponding Image 5 software.
Gel doc ez instrument
Manufactured by Bio-Rad
Sourced in United States
The Gel Doc EZ instrument is a compact and easy-to-use imaging system designed for visualizing and documenting DNA, RNA, and protein gels. It captures high-quality images of stained gels using a CCD camera and UV transilluminator. The Gel Doc EZ system provides a simple and efficient way to capture and analyze gel images for various applications in molecular biology and biochemistry laboratories.
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Lab products found in correlation
3 protocols using gel doc ez instrument
1
RecA-Mediated Strand Displacement Assay
2
DNA Electrophoresis on Agarose Gels
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DNA Electrophoresis on agarose gels was as previously described [26 (link),45 (link)], in which 1 to 1.5% agarose was used in 1 X Tris-acetate- EDTA buffer (1X TAE). We added equal volumes of bacterial supernatants to the wells in each gel experiment, usually 25 µL of the sample mixed with 10 X loading dye. Fluorescent DNA bands on gels were visualized using SybrSafe dye and photographed using the Gel Doc EZ instrument (Bio-Rad, Hercules, CA, USA) and the Image 5 software. Quantitation of gel bands and gel lanes was performed using the Un-Scan-It Gel program for the MacIntosh by Silk Scientific (Orem, UT, USA).
3
Native Gel Electrophoresis of DIX Filaments
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Purified DIX filament and DAX samples were mixed at the indicated concentrations for 30 minutes at room temperature. To each 15 μL mix, 5x native loading buffer (250 mM Tris-HCl, pH 6.8, 50% glycerol, no loading dye) was added. Samples (14 μL) were loaded into lanes where indicated of an Any kD Mini-PROTEAN TGX 15-well gel (#4569036, Biorad, Hercules CA). Electrophoresis was carried out in native Tris-glycine buffer (25 mM Tris, 190 mM glycine, pH~8.3) at 4°C at 25V for 12 hours followed by an additional 2 hours at 100V. DIX filaments labeled with AlexaFluor-488 C2 maleimide (with some residual dye left over from using a 2 mL 7,000 MWCO Zeba spin desalting column) were visualized using the SYBR Green setting of a Biorad Gel Doc EZ instrument with a 10s exposure. To confirm specific detection of DIX fluorescence, DAX samples were run on SDS-PAGE and were visualized using the same acquisition and image contrast settings. Both native-and SDS-PAGE gels were stained with Coomassie Blue for total protein.
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