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What was the collection cloning strategy for the HA-tagged collection?

The cloning strategy is described in the paper for this collection: P. Ross-Macdonald, et.al., Nature 402, 413 (1999). A brief overview is: 1) Mini-transposon integrated into a small piece of yeast genomic DNA that was cloned into a vector at the NotI sites a. The mTn at this point was fairly large containing cre/lox sites, LacZ, URA3, tet resistance, and a few other elements 2) Vector was prepped and digested with NotI to pop-out yeast genomic DNA with mTn integrated in 3) Piece of yeast DNA (with mTn) was transformed into Y800 (diploid) strain of S. cerevisiae 4) mTn integrated into yeast genome through homologous recombination 5) Screened for integrants a. Used URA3 selection (+ strains died, but the lab had duplicates so they knew which colonies were +) b. Also performed B-gal assay 6) Sequenced (+) strains (more accurately the E. coli equivalents of the + strains) to see where, exactly the mTn went into the genome 7) Induced cre-lox recombination in (+) yeast strains to reduce mTn to a smaller, 93 codon insertion a. The mTn at this point was reduced to an Xa protease site and 3X HA. 8) URA3 selection again and if cells lived then URA3 gene no longer there (meaning strain was + for the smaller mTn) There is no special selective media for these. The references for this collection are: P. Ross-Macdonald, et.al., Nature 402, 413 (1999). A. Kumar, et.al., Genes & Development 16, 707 (2002). Kumar, A., Cheung, K.-H., Ross-Macdonald, P., Coelho, P.S.R., Miller, P., and Snyder, M. Nucleic Acids Res. 28, 81 (2000).

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