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Molecular Biology Take-home 1 Due March 1 Midnight A sample from the planet Mars has yielded a new eukaryotic organism, the MARS Amoeba (MARSA)

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Molecular Biology Take-home 1

Due March 1 Midnight

A sample from the planet Mars has yielded a new eukaryotic organism, the MARS Amoeba (MARSA). Initial studies have determined it contains a nucleus with 3 distinct DNA based chromosomes. You have been assigned the task to study this new creature.

Using your knowledge, you are expected to: (25 points each)

1) Generate both MARSA genomic and cDNA libraries while testing your libraries for completeness and titer by using many techniques discussed in our lectures such as Southern and Northern Blotting, mRNA to cDNA techniques, genomic DNA isolation via Principle of pulse-field gel electrophoresis (PFGE) followed by partial restriction digestions, and ligations into lambda phage vectors (expression-based Lambda gt11.).  Include in your written journey all need steps and controls.

2) Use both libraries for a gene screening, clone purification, and characterize these libraries to find the genomic and cDNA clones of three human gene homologues of the RAS/MYC/RB oncogenes.).  Include in your written journey all need steps and controls.

3) Purify these MARSA proteins followed by generating antibodies to these 3 MARSA proteins using techniques including WESTERN Blotting, ionic/size exclusion, affinity chromatography, 1D and 2D SDS PAGE).  Include in your written journey all need steps and controls.

4) Determine Protein Sequences for these new MARSA genes using Edman degradation and Tandem Mass Spectrometry for Peptide and Protein Sequence Analysis).  Include in your written journey all need steps and controls.

5) Use 2Hybrid/Phage Display to characterize the new MARSA genes, their proteins and protein partners).  Include in your written journey all need steps and controls.

6) Apply PCR, Inverse PCR, Differential Display and Real-time PCR to explore the nature of these MARSA genes and proteins).  Include in your written journey all need steps and controls.