Category: DNA Analysis

Preparing a culture of plasmid-harboring Escherichia coli is the starting point in all plasmid isolation protocols. Suboptimal culture conditions and…

A small-scale plasmid isolation procedure, the miniprep, yields a sufficient amount of plasmid for the screening of clones by restriction…

The alkaline lysis method selectively purifies plasmid DNA from other cellular components of the bacterial cells including chromosomal DNA. Controlled…

Alkaline lysis method of plasmid isolation was originally developed by Birnboim & Doly (1979). In this procedure, bacteria harbouring the desired…

Large-scale isolation of plasmid requires a large volume of E. coli culture. Large scale plasmid isolation procedures are termed, midiprep (25 – 50 ml starting culture volume) and maxiprep (100 – 500 ml starting culture volume). A starter culture is initially prepared by inoculating a colony in a small volume (2 – 10 ml) of culture medium. Large culture volume is prepared by diluting starter culture in a ratio of 1: 100 to 1: 1000 in the growth medium.

Bromophenol blue and Xylene cyanol are the two most commonly used tracking dyes for the analysis of DNA on agarose gel electrophoresis. These negatively charged dyes not only help in monitoring the progress of agarose gel electrophoresis but also allow easy monitoring of sample loading process onto the wells of agarose gel. Their position in relation to DNA fragments is an important information that helps to determine how far a gel must be run without accidentally letting the DNA fragments of interest run out of the gel and at the same time ensuring good resolution among different size DNA fragments.

Most of the commonly used plasmid vectors carry at least one antibiotic resistance gene that allows plasmid-containing bacterial cells to…

There are many nucleic acid staining dyes that can be used to visualize DNA embedded in the agarose gel. These…

Overview:
Small-scale plasmid isolation, called plasmid miniprep, requires a small amount of bacterial culture. Normally, a 2 – 5 ml culture provides sufficient cells that can be processed for plasmid isolation using the miniprep protocol. Culture can be prepared by inoculating 3 ml antibiotic-containing growth medium with a single colony and growing it overnight at 37C with shaking.
Here we have taken an example of preparing liquid culture from a colony of E. coli DH5α, transformed with the pEGFP plasmid. The pEGFP plasmid contains a kanamycin resistance gene, therefore, requires kanamycin for the selection of plasmid-containing bacteria. If your plasmid carries another antibiotic-resistant gene, add the respective antibiotic to the culture medium.

Overview: Resuspension buffer is used to resuspend the harvested bacterial cells from the culture during plasmid isolation by the alkaline…

Overview The neutralization solution (solution III) is used for the isolation of plasmid DNA by the alkaline lysis method. The…

Overview Lysis solution (solution II) is used for the isolation of plasmid DNA by the alkaline lysis method. The plasmid-containing…

To isolate plasmid from the host bacteria, cells are first lysed that lead to the release of plasmid and in subsequent steps, the plasmid is purified from the lysate. Purification of plasmid from the lysed cells are mostly dependent on the type of lysis method used to release plasmid in solution. For example, alkaline lysis which completely disrupts the bacterial cells leading to the release of cell components including both plasmid DNA and genomic DNA in denatured state relies on selective renaturation of only plasmid DNA in a perfect manner at the purification step. On the other hand, boiling lysis selectively releases only plasmid DNA from the bacterial cells. The purified plasmid can be further purified by a number of methods to obtain high quality plasmids. These methods are centrifugation in gradients of CsCl – ethidium bromide (EtBr), selective precipitation in high salt SDS, extraction with Phenol-chloroform, and hydroxylapatite and ion-exchange chromatography.

Phenol-chloroform extraction can be used to isolate and purify nucleic acid (DNA and RNA). It is a very reliable method…

Amplification of plasmid is desirable for many applications including gene cloning, DNA sequencing, transfection, and probe preparation. The fastest and routinely used method to amplify plasmid is to introduce plasmid in an appropriate strain of E. coli e.g., DH5α (the process is called transformation), grow them to a suitable culture volume, and finally, extract plasmid from them (the process called plasmid isolation).