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DNA / RNA Manipulation and Analysis

Bacterial Transformation Troubleshooting for Beginners

The first time I did a transformation was when I worked with site directed mutagenesis. I cloned a protein sequence into the p15TVL vector, created my mutants (but that’s another story), and was finally ready for the next step: transformation and expression of my desired protein. Little did I know that my enthusiasm would fall…

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How UV Radiation Causes DNA Mutations

We all know that we are supposed to put on sunscreen in the summer months to protect ourselves from skin cancer, and the connection between sun exposure and cancer is well documented (Koh et al., 1996; Armstrong and Cust, 2017). UV-A and UV-B rays from the sun interact with the DNA in our skin and…

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Restriction Enzymes: Five Things to Consider Before you Chop!

The use of restriction enzymes to characterize DNA has been popular since the 1970s. Today, this “old school” technique is still one of the easiest and fastest ways to assess DNA sequences. Like most lab reagents, restriction enzymes can be fickle and you should bear a few things in mind when using them. Generally, sticky-ended enzymes have greater…

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DNA Extraction from FFPE Tissues for NextGen Sequencing

Rapid genomic analysis offered by next generation sequencing (NGS) is ideal for personalized medicine approaches to clinical genetics, microbiological profiling, and diagnostic oncology. Many standard clinical samples are preserved as formalin-fixed, paraffin-embedded (FFPE) tissues, which presents obstacles for use in NGS analysis. FFPE tissue preservation has the benefit of keeping samples intact for histological examination…

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Those Site-Specific Recombinases in Your Tool Kit

Most of us are aware of genetic engineering systems like Cre-Lox, TALENs, Zinc finger systems, and of course, CRISPR-Cas9.  These are all examples of CSSR- Conservative Site-Specific Recombination. We use these site specific recombinases routinely, but do we really know about them or what the future hold for these tools? It turns out that CSSR…

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Using Synthetic DNA For Long Term Data Storage

The amount of data requiring long-term storage is growing and accelerating. Current long-term digital storage technology cannot keep up. Imagine roughly 2.5 QUINTILLION bytes of data being created everyday in this world1–2 as more computers and network infrastructure come online. For average users, a long-term storage solution is probably not an issue. However, organizations and…

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How to Design a CRISPR Experiment and Start Genome Editing

CRISPR and the CRISPR Associated system (Cas) are powerful gene editing technologies. Originally identified and characterized in bacteria, the endogenous CRISPR systems act as an RNA-based defense mechanism against invading phage DNA. CRISPR cas9 gene editing was adapted for genome editing in 2013 and has since been exploited for its ability to generate targeted double-stranded DNA…

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Transfection Toolkit

Engineering a mutation or overexpressing a recombinant protein to study and characterize its function in mammalian cells is no easy task. Luckily, Chinese hamster ovary (CHO) cells, which have been a mainstay in the lab since the 1950s, represent a relatively easy mammalian model system to engineer. There are several methods to choose choose from…

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Get Your Polymerase Cycling Assembly Oligos Together

The polymerase chain reaction (PCR) is the backbone of many lab techniques. In short, it allows for the exponential amplification of a specific segment of DNA. Through the use of primers encoding restriction enzyme sites, these amplified fragments are used in downstream cloning procedures, usually leading to the insertion of one, maybe two, PCR fragments…

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Quantifying & Assessing RNA: TE or not TE?

Red Pill or Blue? Carrying out science often involves many difficult decisions! I see it all the time in RNA protocols – the “gracious” option of using purified water or Tris-EDTA (TE) buffer to dissolve (or elute, if you are using column purification) RNA. When I was trained in assessing RNA using UV spectrophotometry, graduate…

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Four Ways to Get CRISPR Reagents Into Your Cells

Do you need to test the effects of mutating a gene in your system? Then, CRISPR genome editing is the way to go. Your first step is to decide on good target sequences. Then, you have to get the two components of CRISPR: the Cas9 nuclease and “guide” RNA (gRNA). Even though you’ve read up on…

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Cloning Large or Complex DNA Fragments

Sometimes you know a project is going to be a pain before you even start it. For me, that is whenever I need to clone large (> 3 kb) or complex (e.g., a sequence with repeats) DNA fragments. Long and complex DNA fragments are more likely to create challenges during cloning. Such projects require extra care in just about…

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How DNA Extraction Is Different in Plants

I know what you are thinking, everything is made of cells, so how different can DNA extractions be in plants? The answer is… sort-of different. The overall concept is the same. Cell membranes are lysed, DNA is separated from other cell materials, washed a few times, and then resuspended in water or Tris-EDTA buffer (aka…

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CRISPR Technology Explained: Towards a CRISPR Genome!

If you are working in the biomedical research field and you haven’t yet heard about CRISPR Cas9 gene editing technology, you have some catching up to do – but don’t worry, this article will bring you up to speed about CRISPR in no time! In the few years since the CRISPR phenomenon emerged, this technology has…

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The Three Ts of Introducing Foreign DNA: Transfection, Transduction, and Transformation

Introducing foreign DNA into bacterial or eukaryotic cells is a common molecular biology technique. Some of the terminology involved can be confusing: transformation, transfection and transduction. What do these words actually mean? To make matters worse, sometimes people use these terms interchangeably, resulting in confusion between colleagues and collaborating labs. Here then is your definitive guide…

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Viral Vector Production: Myths & Misconceptions

Viral vector production is a worthwhile skill that can be made even easier with a few tips and tricks. In general, transfection of multiple plasmids into a producer cell line results in infectious, non-replicative virus. However, it is important to ensure that your vector preparation is efficient, giving your experiments the best chance of success.…

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