BC036 – Cloning TRIM6v2 into pGBKT7 via DNA Ligations

Submitted by Brittnee Crane

Hannah Sparks performed a ligation reaction for pGBKT7-TRIM5α and pGBKT7-TRIM6(variant 2) in her experiment HS042. I unsuccessfully completed bacteria transformations with those ligation reaction mixtures. Dr. Graff made a newer (one dot) batch of homebrew bacteria (E. coli C2987), Katie Capp redid the ligation reactions and I completed the bacteria transformation again. pGBKT7-TRIM6(variant 2) (1:4) grew two colonies, while the other plates mention in my lab write-up didn’t contain any visible colonies. Dr. Graff started 2-cultures for each colony that appeared; the cultures from colony #2 grew. I used a GeneJet plasmid purification kit to obtain purified plasmid from the bacteria samples. The concentrations were tested and Katie Capp performed a digestion reaction to verify the insert, which seemed to be what we expected. TRIM6(variant 2) seems to have been successfully ligated into pGBKT7. 

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