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Posts Tagged ‘qPCR’

Oligo Purification Methods: How, Why and for What?

Who amongst us hasn’t had the need for oligonucleotides in an experiment? It is a cornerstone in many procedures and techniques. Depending on the goal, it can be very hard to design just the right oligo for your experiment.  Oligos must have the right length; the right amount of C-G, T-A; they can’t form secondary…

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How to Choose the Correct Reverse Transcription Method

Quantitative Reverse transcription PCR (RT-qPCR) is frequently used in the lab to detect and quantify RNA expression in a sample. The first step of the assay is to convert the labile RNA to its complementary DNA (cDNA) counterpart through reverse transcription (RT). In fact, RT is the first step in a variety of molecular biology…

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Reverse Transcription: The Most Common Pitfalls!

Good quality starting material is king for reverse transcription! Obtaining reliable results in any experiment requires good preparation. We often take reverse transcription for granted, and we don’t always consider that our qPCR might be performing poorly because of problems in that step. Since it’s quite often the reverse transcription reaction itself that causes fuss…

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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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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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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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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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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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10 Tips for Consistent Real-Time PCR

Real-time PCR is a specialized technique that delivers far more information about your DNA or RNA than end-point PCR. Essentially, real-time PCR is a way to visualize the amplification of specific DNA fragments as it is happening (in real time) and allows for the ability to quantify exactly how much DNA (or RNA) was in…

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Are You Ready for Your First qPCR?

Hope you had a heavy breakfast, because the first qPCR is going to take time. Did you remember to fill your coffee cup? There are a lot of intricacies in qPCR that will need your neural networks to be brisk. How qPCR differs from traditional PCR Unlike traditional PCR, qPCR measures the amplification of DNA…

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What Is a Ct Value?

When conducting real-time PCR, you’re looking for the exact amount of a target sequence or gene in your sample. During the PCR reaction, you measure its progress by accumulation of a fluorescent signal during amplification. But there’s also a lot of background fluorescence, which you want to bypass in order to glean meaningful information from…

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Designing Luck: 8 Basic Concepts for Designing Primers for a Standard PCR

I think we all have been through those my-PCR-product-didn’t-get-amplified days. Sometimes, playing around a bit more with the PCR-conditions brings luck, or sometimes it doesn’t work at all. These days we have access to many different types of DNA polymerases, ultrapure and buffered nucleoside triphosphates, and other necessary starting materials in convenient concentrations; but still…

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Quantifying Your NGS Libraries

If you want to get the maximum yield and quality from your next-generation sequencing experiment then you are going to need to make sure each of the libraries you produce is carefully quantified ready for pooling and/or loading onto a flow cell. If the quantification goes wrong you’ll get a bad balance of samples within…

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Mediator Probe PCR

Want a PCR method that gives you high sensitivity and is cost-effective? Then you might want to try Mediator Probe PCR.  This method gives you sensitivity and limit of detection comparable to that of dual-labeled hydrolysis probes without the high cost. We all know how expensive small batch probe synthesis orders can be, especially when…

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