Category: Molecular Biology Lab Notes

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…

In contrast to conventional cloning strategies, Restriction enzyme-free cloning (also called Restriction-free cloning) involves no treatment of DNA fragments to…

ATP-dependent ligase enzyme from Bacteriophage T4 Catalyzes the formation of a phosphodiester bond between juxtaposed 5′ phosphate and 3′ hydroxyl…

TA cloning involves base pairing of adenine (A) and thymine (T)  (complementary base pairs) that result in the joining of…

Molecular cloning or DNA cloning refers to a set of methods that involve in generating identical copies of a piece…

High-fidelity thermostable DNA polymerase for error-free polymerase chain reaction. Possess 3′ to 5′ exonuclease activity for proofreading activity Produces blunt-end…

Also known as KOD HiFi DNA Polymerase High-fidelity thermostable DNA polymerase for error-free polymerase chain reaction. Originally isolated from Thermococcus…

High-fidelity thermostable DNA polymerase for error-free polymerase chain reaction. Possess 3′ to 5′ exonuclease activity for proofreading activity Produces blunt-end…

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…

Overview: ♦ Tissues are composed of multiple cell layers that are tightly glued together by extracellular matrix. ♦ Solubilizing all…