Gene Cloning Problem: Insulin Gene Multiplication Mistake

[Raghav Gupta]

Homework Statement

Molecular cloning experiments are carried out to generate multiple copies of a gene of interest. In one such experiment carried out at a student lab, the gene coding for insulin is multiplied in a bacterial cell. In the screening step, the students realized that they have committed a mistake and they observe many transformed and non-transformed bacterial colonies. Explain this situation with appropriate reasoning.

Homework Equations

NA

The Attempt at a Solution

After the restriction enzymes are used, is it that some plastid is not able to recombine with particular gene and the same plastid combine?

 

[Ygggdrasil]

After the restriction enzymes are used, is it that some plastid is not able to recombine with particular gene and the same plastid combine?

There are a number of explanations I can think of, but yours seems reasonable. I would not use the word "recombine," however, as it has a specific definition in biology. Rather, the correct term for cloning would be ligation. It is worth considering which step in the cloning procedure usually prevents recircularization of the plasmid during the ligation step.

 

[Raghav Gupta]

There are a number of explanations I can think of, but yours seems reasonable. I would not use the word "recombine," however, as it has a specific definition in biology. Rather, the correct term for cloning would be ligation. It is worth considering which step in the cloning procedure usually prevents recircularization of the plasmid during the ligation step.

But what mistakes the student have done? It will happen naturally always that some will transform and some not.

 

[Ygggdrasil]

What step in the cloning procedure normally prevents recircularization of the plasmid during the ligation step? That is the step that was likely omitted if your hypothesis is true.

 

[Raghav Gupta]

What step in the cloning procedure normally prevents recircularization of the plasmid during the ligation step? That is the step that was likely omitted if your hypothesis is true.

I don't know that step. I searched ligation in wikipedia but was not able to get the step.

 

[Ygggdrasil]

This wikipedia article tells you the step that prevents plasmid recircularization in sticky end ligation: https://en.wikipedia.org/wiki/Ligation_(molecular_biology)