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DNA / RNA Manipulation and Analysis

Pulsed Field Gel Electrophoresis: Tips and Tricks

Running a pulsed field gel can be exciting. It isn’t often that you get to visualize such large pieces of DNA. However, it can also be extremely frustrating. It isn’t uncommon to wait 24 hours only to find out that your DNA was degraded before you started the run. Then, you have to start all…

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7 Tips for using Magnetic Beads for DNA Cleanup

Whatever molecular biology techniques you use, at some point you will have to clean up your DNA samples to remove things like buffers, contaminants and nucleotides from you precious sample, so that you have perfectly pure DNA for your downstream experiments. Magnetic beads are one DNA cleanup option. They are simple and effective—and their reassuringly…

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5 Things You Should Know About Agrobacterium Binary Vectors

You can create stably transformed plants expressing your gene of interest; be it for the subcellular localization of your protein or simply for the in planta protein expression and purification. Whatever it is, you can do wonders with plant transformation. Sound difficult? It isn’t. Just like there are millions of microbes that interact with us,…

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Pulsed Field Gel Electrophoresis – The Basics

You have probably run a standard agarose gel hundreds of times. They are great for visualizing small DNA fragments up to 10 kb, but what if you want to examine really large pieces of DNA or even whole chromosomes? This is where pulsed field gel electrophoresis (PFGE) comes in! While the equipment required to run…

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How to Plan a Restriction Cloning Experiment In Silico

Restriction cloning, at its core, is quite simple. You simply cut the target vector and insert with the same enzymes, clean digested vector and insert up, ligate the two together, transform the ligated vector and insert into bacteria, and then screen. While getting each of the steps correct can be a bit of a hassle,…

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Choosing the Right E. coli Strain for Transformation

Cloning, purifying, and expressing modified genetic material is routinely done in microbes such as Escherichia coli (E.coli). Relatives of this molecular biology workhorse normally live in the intestinal track of humans. The particular E. coli strain (K-12) that scientists use all over the world was isolated from the feces of a diphtheria patient in 1922.1…

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SAGE Part 2: LongSAGE, RL-SAGE and SuperSAGE

SAGE, or serial analysis of gene expression, is a technique that enables you to digitally analyze the entire gene expression profile of a cell(s). Before this technique, scientists were limited to studying a few gene’s expression at once by a technique called the expressed sequence tag approach. The coolest part of SAGE is you don’t…

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How to Choose Your Method for DNA Extraction from Whole Blood

Over the last few decades, PCR, next generation sequencing and microarray technologies have taken blood-based research to a new level. Modern blood-based application range from DNA fingerprinting, whole genome sequencing, blood banking to liquid biopsy and many more. Regardless of the application, pure, intact, double stranded and highly concentrated DNA extraction from whole blood is…

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Eight Top Tips to Maximize Yield from Whole Blood DNA Isolation

When you perform genomic DNA extraction from whole blood, low yield or low quality DNA can result in many issues. No matter your intended downstream application—qPCR, next generation sequencing, Sanger sequencing, and so on—you need high quality DNA. We’ve made this step-by-step guide to assist you in getting the highest possible DNA yield and quality, and…

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Essential Factors for Successful Gateway Cloning

An efficient cloning system that is in vogue at present is the Gateway® Cloning Technology. Gateway cloning gives researchers the opportunity to easily transfer DNA fragments into plasmids using proprietary recombination sites called “Gateway att sites” and either the LR clonase or BP clonase enzyme mixes. Gateway cloning is efficient, but it still has multiple…

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Demystifying REBASE and NEBcutter

Two years ago, all I knew was third BASE in my baseball field and the cutter ball from the pitcher. Now, I know a lot more about lab-based BASES and cutters: REBASE and NEBcutter. While they sound like baseball terms, REBASE and NEBcutter are tools for working with restriction enzymes. Read on to find out…

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How to Make Your Own Chemically-Competent Cells

I once had the terrible experience of not being able to run an assay because I ran out of commercial stock of transformation-competent Escherichia coli (E.coli). From that day, I learned to make my own chemically-competent cells in the lab. I recommend that everyone makes their own stash of transformation-competent E.coli stocks—among other suggested laboratory…

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How to Improve Plasmid Yield Using Antibiotics

After the initial excitement of growing and isolating plasmid DNA, routinely preparing plasmid mini/midi/maxi preps gets boring. You definitely want a way to squeeze the maximum amount of plasmid DNA out of your culture. Good news—you can increase your plasmid yield using antibiotics. Keep the Pressure On to Improve Plasmid Yield Remember that you need…

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Polysome Profiles: What You Need to Know

Has your supervisor asked you to do a polysome profile? Are you attempting them but running in to problems? This article will help explain what they are, the basics of the protocol and provide some helpful tips (including some things that I wish I had known when I first started out). Polysome Profiling: The Basics…

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Antibiotics Used in Molecular Biology

Antibiotics are used in a wide range of techniques in molecular biology. My aim with this post is to provide an easy reference to some of the main ones used in molecular biology, their mechanisms, range and working concentrations. I hope you will find it useful. Your Personal Antibiotics Reference Guide *Abbreviations: Gm(+/-)=Gram positive/negative; My=mycoplasma;…

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E.coli Electroporation vs Chemical Transformation

This is the first in a three part series on the transformation of E.coli. By the end of this you should be an expert on E.coli transformation and on which strains to choose for different applications. If you’re already an expert, I hope it’ll be an enjoyable refresher for you. In either case, please comment…

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A Guide to Solid Phase Reversible Immobilization

  Scientists today depend heavily on many molecular biology techniques to perform their research. For example, with the advent of next generation sequencing (NGS): scientists are able to look at very minute details, right down to individual genetic sequence variations. However, the increase in experimental complexity means that every extra step becomes more crucial than…

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