BamHI R6025

Protocol tips

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	Both coding sequences were excised with HindIII and BamHI (Promega). The vector pcDNA3.1+ (Invitrogen) was digested with HindIII and BamHI. The digested components were gel-purified and ligated overnight at room temperature using T4 DNA ligase (Promega).

Protocol tips
Both coding sequences were excised with HindIII and BamHI (Promega). The vector pcDNA3.1+ (Invitrogen) was digested with HindIII and BamHI. The digested components were gel-purified and ligated overnight at room temperature using T4 DNA ligase (Promega).

Publication protocol

The RORα coding sequence was excised from pSport6-RORα, and the REV-ERBα coding sequence was excised from p3×FLAG-REV-ERBα. Both coding sequences were excised with HindIII and BamHI (Promega). The vector pcDNA3.1+ (Invitrogen) was digested with HindIII and BamHI. The digested components were gel-purified and ligated overnight at room temperature using T4 DNA ligase (Promega). The pcDNA3.1+ constructs contain a T7 promoter that allows for in vitro transcription and translation. The TNT kit for in vitro transcription and translation was used to produce protein for EMSA, according to the manufacturer's instructions (Promega). DNA containing putative response elements was annealed and then labeled with [α-32P]dATP using Klenow enzyme (Promega). Binding reactions were prepared using binding buffer (Promega), labeled probes, and equal volumes of protein. Unlabeled probes were used in 10-, 50-, and 100-fold molar excess in competition experiments. Binding reactions were loaded onto pre-cast 5% TBE gels (Bio-Rad) and analyzed by autoradiography.

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Manufacturer protocol

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