Download - Yeast two hybrid screening of liver and WAT libraries I may need 15 instead of 10 minutes
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
Library cDNA recombination in AH109 yeast
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.
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.
PCR identification of clones
250 Yeast plasmids were amplified with PCR and sequenced
Liver clones
Liver clones
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),
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 lipaseNuclear protein SkiP (Ski-interacting protein)profile/Regulator of chromosome condensation containing proteinRho GTPase activating protein 17 (Arhgap17), sex comb on midleg homolog 1 (Scmh1),
syntaxin 5A (Stx5a),
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.