Cloning BRD4 long isoform into overexpression vectors for stable overexpression of BRD4-L in mammalian cells

Abstract

Summary: Molecular cloning of BRD4-L is a challenging technique, because the DNA insert is formed by a long, GC-rich sequence, which folds into secondary structures. The present protocol defines a specific strategy to amplify BRD4-L, followed by the successful cloning of the gene into an overexpression vector. Since there are no existing protocols nor commercially available plasmids, this work provides a useful tool for studies involving molecular cloning of BRD4-L and could potentially be applied to other challenging genes. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• Cell biology
• Molecular biology
• Protein biochemistry
• Protein expression and purification