BC044 – pGADT7 Digests to Prepare for Recombination Reactions

Submitted by Brittnee Crane

This experiment was focused on digesting pGADT7-empty with EcoRI-HF and BamHI-HF so the vector could be used for recombination reactions. It was decided that using pGADT7-T-antigen would be a better choice because the cut-out insert would be visible with gel electrophoresis. There were some complications with band size differences between samples that contained identical plasmids, there was also no inserts visible. The conclusion was that further analysis of pGADT7-T-antigen needs to occur. pGADT7-TRIM5α was digested with EcoRI-HF and BamHI-HF, gel electrophoresis was performed and the pGADT7 band was excised from the gel. The band was put through a gel purification protocol to obtain a purified plasmid that was then ran on a gel. The concentration of the vector was estimated by comparing the band to the ladder.

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