KONSTRUKSI TRIPLE DISRUPTAN GEN PENGKODE PROTEIN FOSFATASE DAN PROTEIN KINASE Saccharomyces cerevisiae
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Abstrak
Konstruksi triple disruptan pada Saccharomyces cerevisiae dibuat dengan menyilangkan (crossing) atau melakukan mating antara strain BY4739 (MATa ura3D0 leu2D0 lys2D0 kin3D::KanMX) dengan strain SH6793 (MATa ptp2D::CgHIS3 msg5D::CgLEU2 ura3-52 his3-Δ200 leu2Δ1 lys2Δ202 trp1Δ63). Dari 10 aski yang menghasilkan 40 koloni triple disruptan, hanya 3 koloni yang memiliki fenotip dapat tumbuh di media SC-his, SC-leu, dan YPDA + 100 µg/mL geniticin disulfat. Uji lanjut terhadap tiga koloni tersebut menggunakan amplifikasi PCR dan pemotongan dengan enzim restriksi NruI menghasilkan hanya satu koloni yang memiliki ptp2D. Data ini mengindikasikan bahwa kemungkinan hanya satu koloni yang memiliki triple disruptan ptp2D msg5Dkin3D yaitu koloni 7B.
Kata kunci: triple disruptan, Saccharomyces cerevisiae, metode crossing (persilangan)
Abstract
Triple disruptant were contsructed by crossing or mating between strain BY4739 (MATa ura3D0 leu2D0 lys2D0 kin3D::KanMX) and strain SH6793 (MATa ptp2D::CgHIS3 msg5D::CgLEU2 ura3-52 his3-Δ200 leu2Δ1 lys2Δ202 trp1Δ63). Out of 10 asci generating 40 colonies which have triple disruptant, only 3 colonies showed phenotypics growing on SC-his, SC-leu, and YPDA+ 100 µg/mL geniticine disulfate medium. Further test to these three colonies by using PCR amplication and digesting by restriction enzyme NruI resulted only one colony showing ptp2D. This data indicated that only one colony had ptp2D msg5D kin3D triple disruptant, colony 7B.
Keywords: triple disruptant, Saccharomyces cerevisiae, crossing method
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