BC030 – Subcloning TRIM5alpha and TRIM6v2 from pGADT7 into pGBKT7

Submitted by Brittnee Crane

Several experiments took place to prepare for the Y2H transformations. Digests were performed with pGBKT7-p53, pGADT7-TRIM5α, and pGadT7-TRIM6(variant 2). The digests were put through gel electrophoresis and the intended amplicons (pGKBT7, TRIM5α, and TRIM6(variant2) were cut out and put through a gel extraction/plasmid purification kit. The gel purified samples were then run on a gel and the estimated concentrations were used to perform a ligation recombination reaction into pGBKT7. Hannah received the desired amplicons for TRIM8 and TRIM31 [HS028] through PCR, I put the cut-out bands through a gel extraction/ plasmid purification, and ran them with the gel purified samples mentioned above. The estimated concentrations were used for a recombination cloning reaction into pGADT7. Dr. Graff and Katie Capp performed the ligation recombination reaction into pGBKT7 as well as the recombination reaction into pGADT7 and completed bacterial transformations with them. Upon pulling the plates, there weren’t any colonies. I completed the transformation again and two of the plates contained colonies, pGADT7-TRIM8 and pGADT7-TRIM13. The colonies were used to seed cultures that were then put through a miniprep plasmid purification, digested with the correct enzymes to ensure that they were what was expected, and the digests were run on a gel. Strange results came from the gel electrophoresis with the digests, so a couple of experiments were performed in attempts to determine why. It was concluded that we had purified bacteria genes. Later we discovered that our SOC media (used in bacteria transformations) was contaminated.

Correction: On page 6 of the attached images, experiment BC031 (rather than BC032) should have been referenced.



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