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Chapter 12: Problem 11

Describe an experimental protocol for the preparation of two cDNA libraries, one from anaerobically grown yeast cells and the second from aerobically grown yeast cells.

Short Answer

Expert verified
The protocol involves growing yeast cells under anaerobic and aerobic conditions, extracting their RNA, converting this to cDNA via reverse transcription, cloning the cDNA into plasmids to create libraries, and validating these libraries through sequencing.

Step by step solution

01

Cultivate Yeast Cells

Yeast cells need to be cultivated under the two different conditions separately - anaerobic and aerobic. The cells should be grown until they reach their exponential growth phase where their metabolic activity is at its highest.
02

Isolate Total RNA

After growth, the yeast cells are harvested and total RNA is extracted. This can be accomplished with RNA extraction kits available commercially. Care must be taken during this step to prevent RNA degradation.
03

Reverse Transcribe RNA to cDNA

This stage involves synthesising complementary DNA (cDNA) from the RNA using an enzyme called Reverse Transcriptase. The enzyme essentially 'rewrites' the RNA into cDNA.
04

Create cDNA Libraries

Now the cDNA, that represents our mRNA profile from yeast cells, can be cloned into a plasmid vector. This is done by incorporating the cDNA fragments into the plasmid's DNA strand, creating a cDNA library. This library is a collection of all the cDNA variants from our yeast cells.
05

Validate the cDNA Libraries

Validate the created cDNA libraries by randomly selecting clones and sequencing them. This ensures that a good coverage of different transcripts was obtained and that the library is representative of the mRNA profile of the yeast cells under both conditions.

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Most popular questions from this chapter

Combinatorial chemistry can be used to synthesize polymers such as oligopeptides or oligonucleotides. The number of sequence possibilities for a polymer is given by \(x^{y}\), where \(x\) is the number of different monomer types (for example, 20 different amino acids in a protein or 4 different nucleotides in a nucleic acid) and \(y\) is the number of monomers in the oligomers. a. Calculate the number of sequence possibilities for RNA oligomers 15 nucleotides long. b. Calculate the number of amino acid sequence possibilities for pentapeptides.

Search the National Center for Biotechnology Information (NCBI) website at http://www. nchi.nlm. nih.gov/sites/entrez?db=Genome to discover the number of organisms whose genome sequences have been completed. Explore the rich depository of sequence information available here by selecting one organism from the list and browsing through the contents available.

Imagine that you are interested in a protein that interacts with proteins of the cytoskeleton in human epithelial cells. Describe an experimental protocol based on the yeast two-hybrid system that would allow you to identify proteins that might interact with your protein of interest.

In your experiment in problem \(12,\) you discover a gene that is strongly expressed in anaerobically grown yeast but turned off in acrobically grown yeast. You name this gene nox (for "no oxygen"). You have the "bright idea" that you can engineer a yeast strain that senses \(\mathrm{O}_{2}\) levels if you can isolate the nox promoter. Describe how you might make a reporter gene construct using the nox promoter and how the yeast strain bearing this reporter gene construct might be an effective oxygen sensor.

Yeast (Saccharomyces cerevisiae) has a genome size of \(1.21 \times 10^{7}\) bp. If a genomic library of yeast DNA was constructed in a vector capable of carrying 16 -kbp inserts, how many individual clones would have to be screened to have a \(99 \%\) probability of finding a particular fragment?
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