MCB473 ModuleIV Lab Report

MCB473 ModuleIV Lab Report - Possible Interaction of...

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Possible Interaction of YDL089W in the Spindle Checkpoint Pathway as Regulatory Kinase of the Anaphase Promoting Complex Written By: Michael A. Miller Investigators: Mike Miller, Kyle Malhotra, Chelsea Baraff, James Achol Teacher: Charles Putnum Sponsoring Organization: MCB 473 Date experimental work was performed: February 27, 2007- April 18, 2007 Date turned in: April 27, 2007 Abstract: YDL089W (herein called “CJM-kinase”) is an identified gene which may manifest functionality in the mitotic spindle pathway. This gene was identified in S. cerevisiae culture as a novel gene in the spindle checkpoint pathway because of its interrelationships with CDC28, CDC37, both with known roles in mitosis. The relationship of CJM-kinase’s involvement in the regulation of the Anaphase Promoting Complex and other functions in the spindle checkpoint pathway is unclear, but may phosphorylate protein complexes crucial to the spindle checkpoint. Introduction:
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Procedures and Materials: Agarose Gel Electrophoresis, AGE Running a gel is used to verify lengths of DNA as well as their concentrations. Under a UV light source, DNA fragments can be observed at different positions in the gel. The smaller fragments move more quickly than the larger fragments. The bands are compared to a “ladder” which is a series of DNA fragments of known sizes. Gels are used to verify the transformation into yeast of the transposon, digestion results, excision of DNA and quantity of amplified DNA by PCR (Module 4, p 4,7,8). Restriction Digests Restriction enzyme digests are used to cut transposon librarys at specific palindromic sequences. The enzyme NotI was used to cut around the transposon in the original vector #36 to verify the transposon was present. Also, restriction enzyme RsaI was used to digest the genomic DNA into smaller base pair lengths (Module 4, p.19 & 30). Transformation Yeast transformation is the process of inserting the vector #36 containing the transposon and library of yeast genes into a host Saccharomyces cerevisiae in order to amplify the transposon library through cell division and budding. Phenotyping is the reason yeast is used over bacteria. It allows screening for mutations to occur. The LiAc and PEG protocol was used for maximum efficiency. The yeast cells were plated onto a minus leucine medium so only the transformed cells with the genetic marker would be able to grow. Eventually, the colonies were inoculated into liquid medium (YPD) and incubated (Module 4, p.20-21). Primary Screen The primary screen was performed on minus leucine, plus galactose plates. In this screen the strain transformed with the transposon library already has within it the GAL-Mps1 construct in- serted into its genome, the phenotype of which is slow growth on GAL. After transforming with the transposon library, LEU2+ colonies are replica-plated to –leu GAL medium. Those colonies exhibiting relief of the growth defect are selected, streaked on –leu plates and retested on GAL. (Module 4, p. 23-24).
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MCB473 ModuleIV Lab Report - Possible Interaction of...

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