Weaver eoc answers 04

Weaver eoc answers 04 - Answers to Weaver end of chapter...

Info iconThis preview shows pages 1–2. Sign up to view the full content.

View Full Document Right Arrow Icon
Chapter 4 Molecular Cloning Methods 1. a) Alu I blunt ends. b) Bgl II 4 bp 5’ overhang c) Cla I 2 bp 5’ overhang d) Kpn I 4 bp 3’ overhang e) Pvu I 2 bp 3’ overhang f) Mbo I 4 bp 5’ overhang g) Not I 4 bp 5’ overhang 2. In order to produce many copies of a DNA fragment we typically replicate it in an organism such as E. coli . Vectors such as plasmids have an origin of replication and by attaching a DNA fragment to the plasmid the foreign DNA will also be replicated. 3. Cloning a DNA fragment into the Pst I site of pBR322 requires that the DNA fragment we want to clone have Pst I sticky ends. We would cut the purified vector DNA with the restriction enzyme Pst I (the Pst I site of pBR322 is within an ampicillin resistance gene) generating complementary sticky ends on the vector. DNA ligase can be used to covalently attach the DNA fragment to the vector and this reaction mixture can then be transformed into E. coli cells. Since there is a tetracycline resistance gene on the plasmid, growing these cells on media containing the antibiotic, tetracycline, will select for colonies carrying the plasmid. Of these tetracycline resistant colonies, the ones which are additionally resistant to ampicillin are ones in which the ampicillin resistance gene has not been interrupted by insertion of a foreign DNA fragment. These contain non-recombinant plasmids. A second sub-group of tetracyline resistant E. coli resulting from the transformation will be ampicillin sensitive. In these bacteria the ampicillin resistance gene will have been inactivated by insertion of the foreign DNA. These are the colonies we want and in order to identify them we can replica plate the entire population of tetracycline resistant colonies onto two media types, the first containing the antibiotic ampicillin and the second lacking the antibiotic ampicillin. The colonies which do not grow on ampicillin are identified and rescued from the ampicillin free plates. These ampicillin sensitive tetracycline resistant colonies contain the DNA fragment we are trying to clone. 4. The multiple cloning site of the vector pUC18 is within a lacZ ’ gene which directs the synthesis of the α peptide of the enzyme β -galactosidase. (The host cell has been engineered to produce the ϖ peptide. Together, the α and ϖ peptides complement each other and produce β - galactosidase activity.) Whether there is an intact lacZ ’ gene in the plasmid can be determined by plating bacteria on the colorless substrate, X-gal, which, when cleaved by β -galactosidase, results in release of a blue dye which will stain the colonies. Interruption of the lacZ ’ gene by a DNA fragment will result in loss of β -galactosidase activity and the growth of white colonies on X-gal. Cloning a DNA fragment into the Bam HI and Pst I sites of pUC18 requires cleavage of the plasmid DNA with Bam HI and Pst I, and ligation of the DNA fragment, which must also have Bam HI and Pst I sticky ends, into the vector. The resulting transformation reaction can be
Background image of page 1

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Image of page 2
This is the end of the preview. Sign up to access the rest of the document.

This document was uploaded on 04/26/2008.

Page1 / 7

Weaver eoc answers 04 - Answers to Weaver end of chapter...

This preview shows document pages 1 - 2. Sign up to view the full document.

View Full Document Right Arrow Icon
Ask a homework question - tutors are online