Category: Molecular Biology Lab Notes

Overview: Cell lines are a very useful tool for life science research. Isolation of total RNA from cell lines is…

Overview: Escherichia coli (E. coli) is one of the well characterized bacteria and is extensively used in both applied and…

Overview: Cell lines are very useful tools for life science research. Isolation of total RNA from cell lines is often…

TRIzol ™  Reagent (Invitrogen)  Supplied by ThermoFisher Scientific  TRI Reagent®  Supplied by MRC, Sigma Aldrich, Zymoresearch RNAzol® (Tel-Test) RNA Stat-60…

Trizol is a very popular reagent among researchers for RNA isolation due to the simplicity and reliability of the isolation…

Overview DNase I is an endonuclease that selectively cleaves phosphodiester bonds of both single- and double-stranded DNA without cleaving RNA….

DNA-free RNA preparations are absolutely required for applications like gene expression analysis using PCR-based methods (e.g., real-time PCR). No RNA…

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).