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Chapter 20: Problem 13

In a control experiment, a plasmid containing a HindIII recognition sequence within a kanamycin resistance gene is cut with HindIII, re-ligated, and used to transform \(E\). coli \(\mathrm{K} 12\) cells. Kanamycin-resistant colonies are sclected, and plasmid DNA from these colonies is subjected to electrophoresis. Most of the colonies contain plasmids that produce single bands that migrate at the same rate as the original intact plasmid. A few colonies, however, produce two bands, one of original size and one that migrates much higher in the gel. Diagram the origin of this slow band as a product of ligation.

Short Answer

Expert verified
Answer: The slow-moving band originates from the ligation of two or more linearized plasmids, end-to-end, forming a larger DNA molecule. This larger molecule migrates at a slower rate during electrophoresis.

Step by step solution

01

Understand the experimental setup

Initially, a plasmid with a HindIII recognition sequence inside a kanamycin resistance gene is cut using the HindIII enzyme. The plasmid is then re-ligated and used to transform E. coli K12 cells. In the next step, kanamycin-resistant colonies are selected, and plasmid DNA from these colonies is subjected to electrophoresis.
02

Analyze the electrophoresis results

In the gel electrophoresis, most colonies contain plasmids that produce single bands migrating at the same rate as the original intact plasmid. However, a few colonies have two bands - one original size band and a slower migrating band. The slower band indicates the presence of a larger DNA molecule.
03

Determine the possible ligation products

Following the HindIII cutting, there are multiple possibilities for plasmid re-ligation: 1. Linearized plasmid forms a circular plasmid by re-ligating its ends. This is the expected outcome (and observed in most colonies). 2. Linearized plasmid with a staggered cut remains open and unligated. 3. Two or more linearized plasmids, end-to-end, ligate together, forming a larger DNA molecule.
04

Identify the origin of the slow band

Based on these possibilities, the origin of the observed slow band could be from the third scenario. This is when two or more linearized plasmids ligate together, end-to-end, forming a larger DNA molecule. This larger size would cause it to migrate at a slower rate during electrophoresis.
05

Diagram the ligation product

The diagram should depict two linearized plasmids, each with staggered cut ends, ligating end-to-end to form a larger DNA molecule. This larger molecule is the slow-moving band observed in the electrophoresis experiment.

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Most popular questions from this chapter

Restriction sites are palindromic, that is, they read the same in the \(5^{\prime}\) to \(3^{\prime}\) direction on each strand of DNA. What is the advantage of having restriction sites organized this way?

A human cDNA clone was isolated from a cDNA library using a homologous mouse gene as a probe. The clone was grown in \(E\) coll, miniprepped to isolate the \(c D N A\), and then amplificd by \(\mathrm{PCR}\) using primers designed to the sequence of the mouse gene. When the PCR reaction was run on an agarose gel, there were multiple weak bands present instead of one clear band. What are the possible reasons for this result? How can the experiment be improved so that one clear band is amplified next time?

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What is a cDNA library, and for what purpose can it be used?
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