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A while back I wrote a post on a T4 DNA polymerase dependent ligation independent cloning method. In the comments, Max asked if anyone had a protocol. Since there does not appear to be a simplified protocol available on the web, I thought I would post mine for reference. It is adapted from a 2006…
Random mutagenesis is an incredibly powerful tool for altering the properties of enzymes. Discover 8 different approaches.
In the early 1950’s so many new enzymes were being discovered in the burgeoning field of biochemistry that enzyme nomenclature was in danger of getting out of hand. With no guidelines on how to name enzymes, researchers simply chose their own. Some enzymes were given names, like diaphorase or Zwischenferment, that conveyed nothing about the…
In any experimental procedure, getting the controls right can save you a lot of work when things go wrong by allowing you to troubleshoot the source of the problem. DNA ligation is no different. In this article, we explain how to set up a ligation reaction with a complete set of controls, and use them…
As a freshman biology major in undergrad, I was introduced to molecular biology with the following description: Molecular biology represents the intersection of genetics, biochemistry and cell biology. Some people, it turns out, add microbiology and virology into the mix. So molecular biology is often used as a catch-all, to describe a wide breadth of…
Few technical breakthroughs have changed the face of their field like the Polymerase Chain Reaction (PCR). Gene cloning, sequencing of complex genomes, DNA fingerprinting and DNA-based diagnostics are just some of the techniques that were either inefficient, crude or plain impossible before PCR. The technique has revolutionized biological research and biotechnology to such an extent…
When heterologous gene expression goes wrong it can be a real headache. Here’s my checklist for the steps to take when you encounter problems with this dark art. 1. Check the construct by sequencing the expression cassette to make sure that everything is as you expect. A lack of expression could result from a stray…
Of all the competent E. coli cell strains available (including both chemically competent or electrocompetent E. coli), which one should you choose? The choice of strain to use in a given experiment is determined in large part by the nature of the experiment and the set of traits that best fit it. In this article…
Buying competent cells from commercial suppliers is convenient, provides a guarantee of quality, and gives access to strains with a variety of in-built traits that assist with things like maintenance of plasmid integrity (more on these traits later). However, this can be an expensive business. Alternatively, competent cells of any strain, including the specially-constructed commercial…
Isn’t it a pain digesting, purifying and dephosphorylating your cloning vector prep to eliminate prevent high background in your ligation/transformation? A new generation of positive selection cloning vectors promises to eliminate all of that hassle by killing off any vector that has not taken up the insert you are trying to clone. Positive selection cloning…
Artificial gene synthesis was first reported in 1972 when a group of researchers at Massachusetts Institute of Technology synthesized a complete yeast alanine tRNA gene. Synthesis of the first peptide- and protein-encoding genes ensued in the following decade. Since then, synthetic biology has advanced in leaps and bounds, and custom gene synthesis, a one-time expensive option for…
Get awesome pictures of your DNA gels with a standard digital camera and an orange filter. Here’s how.
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