Reference no: EM133504668

Molecular and Cell Biology

Practical

Isolation and Restriction Analysis of Plasmid DNA
1. Location
Teaching Laboratory - The Science Place
2. Learning Outcomes
• Understand that plasmids are replicated in bacterial cells
• Describe how a miniprep plasmid extraction works with general reference to underlying chemical principles
• Distinguish between the isolation of plasmid DNA and chromosomal DNA
• Understand the effect and application of restriction enzymes
• Describe the differences in electrophoretic mobility between linear and circular DNA

Introduction
Since the late 1950s and early 1960s, molecular biologists have learned to characterize, isolate, and manipulate the molecular components of cells and organisms. After isolating the first restriction enzyme, HindII, in 1970, and the subsequent discovery and characterization of numerous restriction endonucleases, the Nobel Prize in Medicine was awarded, in 1978, to Daniel Nathans, Werner Arber, and Hamilton Smith. Their discovery led to the development of recombinant DNA technology that allowed, for example, the large-scale production of human insulin for diabetics using Escherichia coli. Over 3000 restriction enzymes have been studied in detail, and more than 600 of these are available commercially.

Plasmids replicate and may express protein within bacterial cells. Naturally occurring plasmids have their own origin of replication and survive by invading bacteria and replicating within the host cell. As simple, independently replicating entities, they provide a convenient means by which a gene of interest can be replicated and expressed in a system small enough that the whole construct can be run on a gel, easily, purified, stored and manipulated.

These natural characteristics of plasmids have been exploited to design plasmid cloning vectors into which a gene can be inserted and the vector can then be transformed into a bacterial cell. In addition to this, the vector can be designed to carry antibiotic resistance to assist in selecting only those bacteria that carry the plasmid after transformation.

Description of practical 2
The plasmid mini prep kit we will be using provides a rapid and economical method for the purification of plasmid DNA in a convenient mini column format. Each column has a binding capacity of at least 20 µg of plasmid DNA. Miniprep columns are designed for use in either a spin or vacuum format. Each column can process up to 4 ml of bacterial liquid culture. The entire procedure can be completed within 20 minutes. The highly purified plasmid DNA can be used immediately for many routine applications such as DNA sequencing, restriction digestion, in vitro transcription, library screening, ligation and transformation.

We will be using Escherichia coli that has been transformed with a plasmid. You will isolate the plasmid from bacterial biomass and chromosomal DNA, purify the plasmid, cut it with restriction enzymes, then electrophorese purified uncut and cut plasmid with given plasmid preparations in a 1% agarose gel.

Experimental protocol
Miniprep Isolation of Plasmid DNA

***Link to protocol PDF is provided in LearnJCU
Restriction Digest
You will be doing one restriction digest. To a labelled microfuge tube, add the following:
• V µl of isolated plasmid DNA (vector map will be provided in LearnJCU)
• X µl of 10x restriction enzyme buffer
• Y µl restriction enzyme (Z units/µl)
• Nuclease free H2O to a total volume of 20µl

Incubate the digest at 37°C for 30 min and then deactivate the enzymes by heating to W°C for T min. Keep V µl of uncut plasmid DNA as a control.

Analyse the DNA on a 1% agarose gel.
*You will have three lanes to load including the DNA ladder, one will produce two bands and the other will give a single band each. Can you explain this in terms of what you have done?*

Agarose gel
You will use the incubation time to prepare your agarose gel. A solution of 1X TAE buffer will be supplied to make your gel and fill the tank with running buffer. You will need to make a 1% W/V agarose gel (1% W/V is defined as 1g of agarose in 100ml of 1X TAE). The gel volume is 30 ml. To visualise the DNA fragments a dye is added (Gelred). The dye stock is a 10,000X concentrate so you will need to determine how much to add to your gel. The demonstrators will guide you through the process of Gel electrophoresis. A 6X times Sample loading buffer is also provided to load your DNA in the wells of the agarose gel. Thus, you will need to determine how much to add to your samples.