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PCR, qPCR and qRT-PCR

Faster PCR Optimization

So, you’ve designed PCR primers to amplify your sequence of interest, and you’re ready to go.  But unless you have a never-ending supply of template, polymerase, and a thermocycler with a gradient function—not to mention a hefty dose of time and patience—you probably don’t want to spend the next week finding the perfect conditions for…

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The Obligate qPCR Standard Curve

At first sight, real-time PCR looks like a very simple technique—very straightforward. Also, when it’s optimized, real-time PCR leads to interesting results. However, to obtain consistent and accurate results reflecting the reality, good controls are crucial for SYBR qPCR.  One of these controls is the qPCR standard curve to check for the efficiency of your primers. Efficient Primers…

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DNA Shuffling Like a Pro

DNA shuffling uses PCR technology in a very creative way. It allows you modify your protein to make a new protein you want. You can evolve proteins in microcentrifuge tubes on your very own lab bench. Isn’t that fantastic? DNA shuffling is also a very powerful technique for directed molecular evolution. W. Stemmer first used…

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From Revolution to Evolution: Stem-loop Real-time PCR

Kary Mullis invented polymerase chain reaction (PCR) in 1985 creating a revolution in molecular biology techniques. But it hasn’t stopped there. PCR has greatly evolved over the years. Today, we stand at a point, where we can clone micro RNAs (miRNAs) in real time! Due to miRNA size (about 18-21 nucleotides long) and varied expression levels,…

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How to Choose the Right Pipette Tips for your Experiment

The precision and accuracy of even the best calibrated pipette can be wiped out if you choose the wrong kind of tips. Depending on the experiment you are doing, the wrong kind of tips can also make your pipette a source of contamination, lead to waste of precious samples or reagents—or even cause you physical…

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The Real-Time PCR Digest

In the 30 odd years since its invention, the polymerase chain reaction (PCR) has become the bread and butter technique of molecular biologists. The secret to its indispensability lies in its simplicity and versatility. Numerous variants of the technique have been developed; one of these, real-time PCR, has become the method of choice for quantitative…

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The Essential PCR Troubleshooting Checklist

Routine PCR? Let’s be honest, there’s no such thing. Even with the simplest PCR reaction things can go wrong, so you need to have a good checklist of ideas for PCR troubleshooting and rectifying the problem. Today I have brainstormed all of the ways I can think of to approach problems with standard PCR reactions.…

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SPUD’s Your Bud When it Comes to Sensitive qPCR

There’s piloting a brand new technique for the first time. Then, there’s jumping through hoops trying to get an established lab technique to work. The former, in contrast to the latter, is expected to be fraught with hardships. Yet troubleshooting an old lab technique that isn’t working anymore, is frustrating at a whole new level.…

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Touchdown PCR: A Primer and Some Tips

When I first heard of touchdown PCR, I thought of a landing aircraft, which, as it turns out is not a bad way to think about it. But despite it’s amenability to analogies and dreadful puns (see title), touch-down PCR (TD-PCR), a very useful technique for improving PCR amplification specificity, is trickier that it might…

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Do You Know Where You Are? Find Out with Genome Walking

Do you work with plants? Are they genetically engineered? Do you know where and how? If not, you could experience problems. After all you do not want your transgenic gene cassette to disrupt genes that would affect your phenotype of interest. In this article I will tell you about Agrobacterium-mediated transformation – a widely used…

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2015 Staff Picks: PCR & Real-time PCR

To wrap up 2015, we’re highlighting our favorite articles from the year. These articles were handpicked by our editorial staff here at Bitesize Bio. So, sit back with your cup of coffee (or beverage of choice) and enjoy these fantastic articles from 2015. First up, we have the category of PCR & Real-time PCR. Jen…

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Long-Range PCR: It’s About Choosing the Right Enzyme

The ability for DNA polymerase to copy a long stretch of DNA is becoming increasingly important. Why? It has to do with the advances in our sequencing technologies. Our next generation sequencing (NGS) technology requires the DNA polymerase to copy a long stretch of DNA (sometimes up to 50kb) as NGS is churning out genetic…

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PCR Problems? Try an Additive

You’ve tried all the usual stuff, and checked the primer sequences twice, but still can’t get that PCR fragment amplified. It’s time to enter the strange world of PCR additives. Over the years a variety of additives have been shown to enhance PCR reactions in certain situations. Here is a summary of some of the…

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How Thermophilic Bacteria Survive, Part II: DNA

In part I, I answered the question, “How do proteins in thermophiles survive under high temperatures?” In this part, I’ll look look at how nucleic acids survive -thrive, even- in conditions that are too hot for most of us, but ideal for a number of organisms, including the one that gave us Taq polymerase and…

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Photonic PCR: When Lightening Strikes Your DNA

Before I get into today’s topic, please allow me to digress a bit and start with a few sentences that sum up the polymerase chain reaction (PCR); the grand-daddy of molecular biology. PCR, a method that is at the heart of modern day molecular biology discoveries, is a process that amplifies genetic material through our…

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Time to Instigate Nested PCR

How to Obtain a Purer PCR Product and Reduce Non-specific Amplification Unless you’ve gotten your hands on some miraculously specific primers, amplification of only your target sequence without non-specific amplification can be very challenging. Thankfully, a clever and surprisingly simple solution is at hand! A Quick Recap of the Basics In PCR, you design your…

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Get that Genotyping PCR to Work EVERY TIME

Say you just joined a lab and have been assigned your very own project to work on. As part of your new responsibilities, you have to breed and maintain the mutant (or transgenic) mouse line which you will be using for your experiments. An integral part of mouse genetics experiments is determining the genotype of…

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