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Describe the method for making cDNA libraries for a yeast 2-hybrid screen.

notes: must draw on the references which i uploaded including the papers, slides and the Y2H manual, and i also upload a sample from others, you can also refer to the sample.The assignment hand via Turnitin, the similarity much less than 10%.

Workshop II. Assessment (word limit 1500 words) via Turnitin
Answer the following questions. The marks out of 20 for each question are indicated. The total word limit is 1500 words – you can decide how much to devote to each question. You should use Figures where appropriate.
1. (4/20) Summarise the paper by van Dongen et al (2004) which describes the use of Y2H to demonstrate the interaction between copper chaperone proteins and the N-terminal metal binding domain/s of copper-transporting P-type ATPases.
Read the Y2H manual, esp. pp. 4-6, 11-13, 15, 17, 19, 21-22, 27, 30, 32-33, 38-39.
Also – see Powerpoint presentation on LMS.
2. (3/20) Describe the method for making cDNA libraries for a yeast 2-hybrid screen. Remember that a library is a set of cDNAs cloned into an appropriate vector.
3. (4/20) Briefly describe the different reporter gene constructs used to select for, or to score, 2-hybrid interactions in yeast. How are they used?
4. (1/20) What is 3-AT and what is it used for?
5. (4/20) You have identified a cDNA clone which activates reporter gene expression in yeast when using a particular bait. What further experiments might you carry out in yeast to verify the protein-protein interaction is truly occurring (in yeast)? What controls are important?
6. (4/20) Having done the above, how can you test that a Y2H interaction that appears robust in yeast is “real”. In other words, what sort of experiment might you do to show the two proteins actually interact in the organism in which the proteins are normally expressed?
Briefly, describe two methods that you could use.
The adequacy of your referencing will contribute to the mark for each question (lack of appropriate references minus 1⁄2 mark).
Word Limit Total word limit: 1500 words. Not including Figures or their legends. A penalty will be applied to work over the word limit


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