MCB 2050 Lecture 1: mcb lec 1

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chocolatelion965

8 Mar 2017

School

University of Guelph

Department

Molecular and Cellular Biology

Course

MCB 2050

Professor

D Mosser

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Document Summary

In order to study the function of an individual gene you must isolate it and produce large quantities of it in vitro. Step 1: fragment the genome into small pieces - cut dna with restriction enzymes. Step 2: create a dna clone library to isolate each fragment. Put fragments in a cloning vector (plasmid) & transform them in e. coli to amplify cloned dna. Each colony has many identical cells, each cell w a copy of the identical cloned dna fragment. Plasmids are small, extra-chromosomal double stranded dna molecules that can replicate in bacteria. Screen colonies using genetic/hybridization techniques & select the colony of interest for step 4. Step 4: amplify plasmid dna for further study. Grow e. coli cells containing the plasmid from a colony in broth culture to amplify the (recombinant) plasmid dna. Isolate plasmid dna from lysed e. coli cells. Analyze the plasmid dna (e. g. size, sequence)

Related Questions

A kanamycin-resistant, ampicillin-resistance plasmid (3 kb) is cleaved with the PstI enzyme, which cuts within the ampicllin resistance gene. The digested plasmid is ligated to a 5-kb PstI DNA fragment from Drosophila (fruit fly). The DNA mixture was then transformed into E.coli host cells.

(i). Which antibiotic would you put into the medium (nutrient agar plate) to select for cells that contain the plasmid?

(ii). Explain step by step, how you would select for cells containing the recombinant Drosophila DNA.

(iii) If different colonies from the plate in part (ii) are cultured in nutrient broth and DNA was isolated from each of the bacterial cultures, what restriction patterns would you expect to see following PstI digestion and agarose gel electrophoresis?

jadehound73

1. We can construct our gene library by cloning the PstI fragments into either plasmid pBR322 or phage lambda. Which one should we choose and why?

2. Suppose we want to insert the PstI fragments into the Bam HI site within the gene for tetracycline resistance (tet^r) in plasmid pBR322. If both of these restriction enzymes produce cohesive ends, how can this be done?

3. After transforming E.coli with the plasmids, how can we identify the cells that contain a chimeric plasmid (containing DNA insert)?

4. If a million tetracycline-sensitive, ampicillin-resistance clones are grown on nutrients agar plates, how are we going to detect the rare clone or clones that carry the gene for protein P?

5. After selecting a clone carrying the gene for protein P, its ells are propagated to high density in nutrient broth. The chimeric plasmid (with an insert of the gene for protein P) is then extracted and purified from the rest of the cellular DNA. How can we now isolate the gene from the plasmid?

6. How can we demonstrate that the gene we have isolate is indeed the one for protein P?

lemonrat870

Recombinant DNA Technology

The questions all relate to the same topic and somewhat interrelate

1. Plasmids used in vitro to clone foreign DNA fragments are called ___

A. Transgenic.

B. Clones.

C. Vectors.

D. Conjugants.

2. VNTR alleles are hypervariable regions of human DNA that differ from each other in:

A. Location of internal sites recognized by restriction enzymes.

B. Variable number of point mutations.

C. Number of copies of an internally repeated DNA sequence.

D. Variable location on different chromosomes.

3. Probability calculations are used in forensic applications of DNA fingerprinting to determine if:

A. DNA from two different sources has matching alleles.

B. DNA samples were degraded before analysis.

C. A match between alleles of different DNA samples might have occurred by chance.

D. An unknown DNA sample is from a male or a female.

4. Plasmid vectors used in cloning often contain a gene for the N-terminal 146 amino acids of the enzyme Î²-galactosidase. What is the purpose of including this gene in the vector?

A. It allows selection of E. coli host cells that contain the plasmid.

B. It allows selection of E. coli host cells that contain plasmid in which the insert has been ligated.

C. It cleaves the insert to allow it to be ligated into the vector.

D. It enables the plasmid vector to replicate in E. coli host cells.

5.The first gene therapy clinical trial took place in 1990, on patients with SCID (severe combined immunodeficiency syndrome). Is this true or false?

A. True

B. False

copperferret659

MCB 2050 Lecture 1: mcb lec 1

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