Week 2 – Brittnee and Kurtiss

Submitted by Brittnee Crane for TRIM Protein Research

May 14th– May 20th 

More yeast colonies were streaked out onto “pizza” plates. `A culture for T6(2)-91 was started for Yeast DNA purification and then the purified plasmid was used in bacteria transformations.  Due to a mistake during purification, the transformation was unsuccessful. Cultures were started for both glycerol stocks and yeast purification with T6(2)-5(1), T6(2)-5(2), T6(2)-44, T6(2)-54, T6(2)-69, T6(2)-70, T6(2)-75, and T6(2)-78.  The culture for #5(1) was unsuccessful. After yeast DNA purification with all the samples, the plasmids were used to transform bacteria and plate them onto ampicillin agar plates. Ampicillin plates were used as all prey plasmids (unknown proteins) were inserted into pGAD, which contains ampillicin. #91 was cultured again.  

 The PCR reactions mentioned in the previous week: TRIMs 6, 6*, 31, 31*, 69, and 69* (with *=1 ng of plasmid as template and without=0.5 ng of plasmid as template), went through gel electrophoresis and were ALL successful!! The desired amplicons were extracted from the gel and purified. Gel electrophoresis was performed with the gel purified plasmid and the concentration was estimated for each. The gel purified plasmids were used along with purified pGBK digested with EcoRI and BamHI, to complete recombination into pGBK. Bacteria transformations took place with the recombination reactions twice and were unsuccessful both times, except for one transformation. PGBK-TRIM6 grew a single colony that was obtained and cultured.  

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