Cloning-DNA Agarose Gel Electrophoresis of Digested Plasmid and Selection for Sequencing | PraxiLabs

Cloning-DNA Agarose Gel Electrophoresis of Digested Plasmid and Selection for Sequencing Virtual Lab Simulation

Biology | Biochemistry | Genetics | Microbiology



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General Aim

To visualize the DNA fragments of the isolated restricted plasmids to be able to choose one for sequencing.

Method

Agarose Gel Electrophoresis.

Learning Objectives (ILO’s)

  • To prepare the agarose gel properly.

  • To identify the concepts of gel electrophoresis.

  • To understand the role of the devices and the reagents used in the processes of agarose gel electrophoresis.

  • To load samples onto the gel.

  • To execute a proper run of agarose gel electrophoresis.

Theoretical Background/Context

The purified plasmids from unit 4 were digested using restriction enzyme digest. In this unit, you will run the restricted purified plasmids on agarose gel electrophoresis to be able to visualize DNA fragments and determine if you were successful in obtaining a recombinant DNA molecule.

Gel Electrophoresis is a procedure used in molecular biology to separate and identify molecules (such as DNA) by size. The separation of these molecules is achieved by placing them in a gel made up of small pores and setting an electric field across the gel. The molecules will move based on their inherent electric charge (i.e., negatively charged molecules move away from the negative pole) and smaller molecules will move faster than larger molecules; thus, a size separation is achieved within the pool of molecules running through the gel. The gel works in a similar manner to a sieve separating particles by size.

 

Principle of Work

DNA possesses negative charges. Thus, DNA moves towards the positive end (anode) when a current is applied across the gel. The agarose contains SYBR Safe DNA Stain which intercalates into the DNA, allowing visualization using a blue light transilluminator.

A DNA size marker is a large piece of DNA that has been digested with one or more restriction enzymes to produce a known array of DNA fragments that range in size. Loading a size marker in the reference lane allows you to determine the approximate sizes of the DNA fragments from your sample.

 


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