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Yeast two hybrid screening of liver and WAT libraries

Yeast two hybrid screening of liver and WAT libraries. I may need 15 instead of 10 minutes. Bait & prey ?. cDNA library inserted here. Library construction. RNA reverse transcription PCR amplification of cDNA recombination of cDNA with pGADT7-rec vector.

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Yeast two hybrid screening of liver and WAT libraries

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  1. Yeast two hybrid screening of liver and WAT libraries I may need 15 instead of 10 minutes.

  2. Bait & prey ?

  3. cDNA library inserted here

  4. Library construction RNA reverse transcription PCR amplification of cDNA recombination of cDNA with pGADT7-rec vector

  5. Library cDNA recombination in AH109 yeast

  6. First screening Cotransformation of pAS2-1-PERK, cDNA and pGADT7-rec Media are deficient of Trp, Leu, Ade, His. ((But His is leaky.)) AH109 yeast contains mutations in TRP1, LEU2, ADE2 and HIS3. AH109 has four reporters—ADE2/HIS3/MEL1/lacZ controlled by GAL4.

  7. Re-streak to separate different plasmids Big clones were picked and streaked 2-3 times on –Ade/-His/-Trp/-Leu media plus 2.5mM 3-AT. The 4th streak was done on –Ade/-His/-Trp/-Leu media plus 7.5mM 3-AT.

  8. PCR identification of clones 250 Yeast plasmids were amplified with PCR and sequenced

  9. Liver clones

  10. Liver clones

  11. Liver clones adaptor-related protein complex AP-1, mu subunit, ATX1 (antioxidant protein 1) homolog 1, BTB domain containing 9 (Btbd9), gamma-aminobutyric acid receptor associated protein (Gabarap) high mobility group 20 B (Hmg20b) microtubule-associated protein 1 light chain 3 beta (LC-3) Mus musculus polymerase, delta interacting protein 2 (Poldip2), protein inhibitor of activated STAT, 4 (Pias4), SNF related kinase (Snrk),

  12. WAT clones cell division cycle 5-like (S. pombe) (Cdc5l), Ewing sarcoma breakpoint region 1 (Ewsr1), G protein beta subunit-like (Gbl), GABA-A receptor-associated protein (Gabarap) HRAS like suppressor 3 (Hrasls3), leukotriene C4 synthase (Ltc4s), lipoprotein lipase Nuclear protein SkiP (Ski-interacting protein) profile/Regulator of chromosome condensation containing protein Rho GTPase activating protein 17 (Arhgap17), sex comb on midleg homolog 1 (Scmh1), syntaxin 5A (Stx5a),

  13. What’s next ? Cotransformation of the postive PCR product back to AH109. GST pulldown ? Co-IP ? Any one passes all the requirement will be further characterized in vivo.

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