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Gene cloning and expression
Specific goal: create and test a set of expression constructs for testing the in vitro interactions between Yar1p (mutant and wild-type versions) and its putative interactors: Rps3p, Ltv1p, and Yor021Cp.
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9/6/00 |
Introduction to Restriction Enzymes and Gel Electrophoresis |
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9/13/00 |
Preparation of yeast genomic DNA |
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9/20/00 |
Design & Set up PCR reactions (may be actually run during week) |
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9/27/00 |
Gel analysis of PCR products, Strataprep PCR purification |
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10/4/00 |
Restriction digests of PCR products and vector, gel purification, ligation set-up |
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10/11/00 |
E. coli transformation (make competent cells and transform lig.s) |
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10/18/00 |
Minipreps of candidates, design analytical digests |
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10/25/00 |
RE digests, gel, conclusions |
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11/1/00 |
Midi prep and phenol extractions, DNA quantitation |
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11/8/00 |
Induction of GST and GST-YAR1, E. coli cell harvesting |
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11/15/00 |
In vitro trsc/trsl with biotin lysine, GST pull-down assays |
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11/22/00 |
Thanksgiving break (no lab) |
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11/29/00 |
Protein gels and transfers |
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12/6/00 |
Western blots (streptavidin for biotin) |
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12/13/00 |
Final paper draft due, discussion, conclusions, revision |
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Tues, 12/19, 8:30 to 11:30 am |
FINAL EXAM |
*subject to revision as the semester proceeds
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