Library Vector

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Library Vector
Bait- cd3d Vector
x
membrane
OR
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cDNA Library DUALMembrane Jurkat T cell cDNA
library in library vector pDSL-Nx. Complexity
9x106 independent clones Average insert size
1.5kb Size range 0.8-6kb Bait vector
pBT3SUC-cd3d (has the SUC signal sequence for
membrane targeting and has a deletion of the
signal sequence of the mammalian cd3d gene
itself) Previous controls Bait expression test
Transform yeast NMY51 with 1.5 mg of constructed
(pBT3SUC-cd3d ) and control (pCCW-Alg5) bait
vectors. Result in SDleu plates too many
colonies, none in other plates(SD-trp,SD-trp-leu,S
D-trp-leu-his,SD-trp-leu-his-ade. Showing that
leu containing bait vector is expressed in yeast
and there is no self activation of bait vector
constructed.
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Bait control assay
Control assay Transform NMY51 with constructed
bait (pBT3SUC-cd3d) and sequentially transform
with positive control prey vector pAI-Alg5(with
Nub) or negative control prey vector
pDL2-Alg5(with NubG).
Showing that there is no self activation of bait
vector and also there is no leakage of HIS or ADE
in the absence of an interaction. Positive
control confirm the presence of bait and
functioning of the Cub portion of bait protein.
Overall results indicate the functioning of the
auxotrophic reporters used.
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Bait control assay
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• Library transformation
• 28mg of cDNA library plasmid was transformed into
  yeast containing bait vector. Transformed yeasts
  spread on 20 x SD-leu-trp-his 150mm glass plates.
  
• Colonies grown on this primary plates (542 col.)
  were transfered to 16 x SD-leu-trp-his-ade 90mm
  petri plates. And b-galactosidase assays were
  carried out via filter lift-off assay.
• Result

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An example of SD-leu-trp-his-ade plate and its
B-gal assay via filter lift-off.
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IDENTIFICATION OF POSITIVE CLONES

• Direct colony PCR from yeast Resuspend yeast
  colonies in 0.02N NaOH ,
• Use as template for PCR. Primers prey sequencing
  primers

Example Colony PCR results
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