DNA / RNA Manipulation and Analysis

MicroRNAs (miRNA) are short, non-coding RNAs involved in post-transcriptional silencing of gene expression. miRNAs can be associated with exosomes and can function as cancer-specific biomarkers. This, coupled with the fact that they are stable in plasma and serum makes them valuable diagnostic tools, as long as they can be reliably isolated from the serum and…

Alternative splicing is a highly orchestrated process that uses a multitude of regulatory mechanisms. Splicing specificity involves a precise interaction between cis- and trans-acting regulatory elements, and factors that disrupt these interactions can result in aberrant splicing. There are multiple ways in which mutations can affect splicing fidelity: A point mutation in the cis-acting splice…

The Basic Local Alignment Search Tool (BLAST) algorithm is at the heart of a free suite of online resources available through the National Center for Biotechnology Information (NCBI).  While most researchers are aware of BLAST as a sequence alignment tool, NCBI’s BLAST suite offers so much more!  I’ll cover in-depth how to use these resources…

Alternative splicing events often occur in a spatiotemporal manner, and some are regulated by alternative splicing regulators, with striking variation across tissue types and developmental stages. Alternative splicing events are often differentially regulated across tissues and during development, as well as among individuals and populations, suggesting that individual isoforms may serve specific spatial or temporal…

The stability of an antibiotic depends on its chemical structure, method of isolation (from natural sources or chemical synthesis), and the mechanisms of inactivation. First generation antibiotics isolated from natural sources, such as penicillin, are the most unstable, followed by its semisynthetic derivatives (such as ampicillin and carboxycylin).  Aminoglycosides (kanamicin, spectinomycin, etc.) are more stable.…

Whether you’re employing sequencing gels, Sanger-based methods, or the latest in pyrosequencing or ion torrent technologies, obtaining, manipulating and analyzing your sequences has never been easier. Depending on what your goals are, you need to understand the pros and cons of the software. There is a lot of software out there, so do you your…

Fortunately, those of us who have learned how to sequence know that aligning sequences is a lot easier and less time consuming than creating them. Whether you’re employing sequencing gels, Sanger-based methods, or the latest in pyrosequencing or ion torrent technologies, obtaining, manipulating and analyzing your sequences has never been easier. We’re going to take…

Collecting biological samples in the field can be difficult, since storage conditions outside of the lab are often less than optimal. Enter the Whatman FTA (Flinders Technology Associates) Cards. The Whatman FTA Card, a filter paper product manufactured by GE Health Care, is a paper matrix laced with a proprietary mixture of chemicals that lyse…

Commercial kits for isolation of large quantities of plasmid DNA generally rely on standard alkaline lysis followed by an affinity chromatography column-based method to purify DNA. Compared to traditional cesium chloride banding or PEG precipitation of plasmid DNA they are a breeze, and have the added benefit of avoiding use of toxic or hazardous chemicals…

Working with RNA? What fun! Those little, nearly indestructible RNases are everywhere – on your skin and mucous membranes, in the water and (some of the) enzymes you use, on lab surfaces, even in airborne microbes! Here are 10 ways to keep the RNases at bay, and keep your precious samples safe:

Microarrays are one of the most in-depth ways of determining cellular gene expression levels of thousands of genes simultaneously.  They are able to help determine: Gene function and cellular processes Gene regulation and  interactions Gene expression levels in different cell types and how this expression is altered by the addition of various compounds or disease…

Most site-directed mutagenesis protocols strongly recommend that you use only PAGE- or HPLC-purified primers to mutate plasmid templates.  Using purified primers is supposed to minimize the introduction of unintended mutations, thus drastically improving the probability of generating your desired mutant.  However, specially purified primers can be extremely expensive, and take longer to synthesize than standard…

Working with RNA is definitely an acquired skill.  It’s a lot more finicky than working with DNA, and requires careful attention to detail in order to avoid disastrous through RNase contamination.  Here are a few common ways to lose your hard-earned RNA:  1. Don’t keep everything on ice Keeping the temperature of all of your reagents cool is…

Restriction enzymes are a basic tool in the molecular biologist’s arsenal.  They’re super easy to use, and virtually essential for cloning and other applications.  Restriction enzymes are also a great example of a perfect “tool” from nature that scientists have co-opted for their own use.  Here are a few fun facts about restriction enzymes that…

Site-directed mutagenesis studies can be extremely useful for elucidating the function of a gene or protein, or for creating variants of an enzyme with new and improved functions. There are now many approaches available for generating site-directed mutants, whatever your purpose. In this post I’ll summarize three techniques that will enable you to produce a…

We recently featured an article about how to streamline your cloning.  But what about those days when you have too much on your plate, and need to put some things off until later?  Here are a few hints on where you can pause in your cloning experiments while working on other projects: Restriction digests can…

I always keep an ear open for helpful tips in the lab – those little tricks that can make your experiments faster, easier and better. Here are a few tricks I’ve picked up for trimming down the time it takes to do your cloning: Restriction digests Many digests are complete within 10 minutes of digestion…

Working at a plasmid repository, I get a lot of feedback from scientists who are relieved we exist simply because that means they don’t have to request a plasmid directly from another academic lab. Either they’ve had a bad experience making requests in the past, or they really don’t know how to go about doing…

TAE or TBE, which is best? Well, of course, it depends on what you want to do. Here are the pros and cons of both: TBE (Tris-borate-EDTA) is a better conductive medium than TAE (Tris-acetate EDTA) so is less prone to overheating so use TBE for long runs Borate is an enzyme inhibitor so TBE…

The following question was emailed to Bitesize Bio by Beheroze Sattha and I gladly took up the challenge, and I immediately knew the answer. Or so I thought. After delving extensively into Pubmed, Genes V (I know, I need a new version) and Molecular Cloning I have come up with an answer, but it is…