Assignment 4

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Lecture 11

• Assignment 4
• Notebook
• Beta-galactosidase assay
• What do you want me to talk about in the review?

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Experiment 3

• Remember you have to prepare in your notebook the
  volumes you are going to use for your restriction
  digestion.

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Assignment 4

• PART 1 Experiment 3
• Page 1 Will present 3 labeled gels 1 the gel of
  the digest of the vector and digest for DNA
  fragment isolation 2 the gel of the ligation
  reactions and isolated DNA fragment 3 the gel of
  the restriction digestions of the two clones.
• Page 2 You will summarize the success or failure
  of each step of the process of cloning.
• PART 2 Experiment 5
• Page 3-4 will be your report on the yeast mutant
  screen. The text following the table shall answer
  four questions about the mutants you
  characterized.

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Summary of Expt. 5 in your notebook
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Notebook

• Look at the instructions and expectations on the
  web site.
• Goal is to have a document that you can show a
  potential employer or supervisor demonstrating
  what you have experience doing and that you have
  the ability to record data in a true functioning
  lab notebook.

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The LacZ Reporter Gene
beta-galactosidase is an enzyme encoded by the
bacterial gene lacZ. beta-galactosidase cleaves
the colorless substrate X-gal (5-bromo-4-chloro-3-
indolyl-galactopyranoside) into galactose and a
blue insoluble product of the cleavage.
Therefore, lacZ can be used as a reporter gene
(blue/white selection)
X-GAL
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Cis-acting elements
URS 1
lacZ
URS 2
GAL1-10
URS 2
lacZ
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Cis-acting elements
Mother daughter
URS 1
lacZ
Late G1 in both mothers and daughters
URS 2
lacZ
Galactose inducible in both mothers and daughters.
GAL1-10
lacZ
Apart the elements are independent together the
elements are restrictive and not additive.
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Properties
URS 1
lacZ
URS 2
Haploid specific Cell cycle regulated Mother
cell specific
GAL1-10
URS 2
lacZ
Haploid specific Cell cycle regulated Galactose
inducible
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Trans-acting factors required for expression
Trans acting factors
Cis element
URS1
Swi5p
Swi1p, Swi2p, Swi3p
URS2
Swi4p, Swi6p
Swi1p, Swi2p, Swi3p
GAL1-10
Gal4p
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Therefore in swi5lf or shelf mutant haploid cells
grown on Galactose plates
URS 1
CAN1
URS 2
Is not expressed
BLUE
GAL1-10
URS 2
lacZ
Is expressed
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Therefore, in the other swi mutant haploids grown
on Galactose plates
URS 1
CAN1
URS 2
Is not expressed
WHITE
GAL1-10
URS 2
lacZ
Is not expressed
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Summary of Expt. 5 in your notebook
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What would you like me to explain to you now, and
what would you like me to review next lecture?