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shoba_anantha

Shoba works at a biotech company in Wisconsin. She has MS from the University of North Carolina.

Articles by shoba_anantha:

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…

09 Jul 2016 PCR, qPCR and qRT-PCR

10 Tips for Pipetting Perfection

Several years ago as a freshman in a research lab, the very first project I received was to pipette incremental micro-volumes of H2O onto a piece of parafilm. Boring! Weighing the liquid on parafilm and comparing the weight between 10 replicates for each micro-volume continued for a week before I touched anything else in that…

28 Nov 2011 Equipment Mastery & Hacks

Eliminate PCR Amplicon (but not real-time PCR?) Carry-Over With UNG

Using UNG is a great trick for PCR amplicon decontamination, but is it any good for rtPCR?

08 Oct 2010 PCR, qPCR and qRT-PCR

PCR: The Right Way to Decontaminate and Eliminate False Positives

PCR is highly sensitive, but the downside of that very property is that it makes the technique prone to producing false-positives. In labs where PCR is a staple, like the one I work in, any false-positives are more often than not due to amplicon contamination. A broken capillary or a PCR plate left carelessly at…

09 Apr 2010 PCR, qPCR and qRT-PCR

How to Build a Plate Centrifuge for $25

I recently visited a lab that had a salad spinner on their lab bench and at first I wondered if they were putting together a salad lunch there but when I took a peek I got a nice surprise. It turns out that the salad spinner was actually a bench top, “minifuge” version of a…

12 Mar 2010 Equipment Mastery & Hacks

qPCR: Plexor vs Hydrolysis Probes

Hydrolysis probes, commonly also referred to as TaqmanTM is a very popular chemistry for real-time PCR. In this article I will compare hydrolysis probes with PlexorTM. But first, a quick overview of hydrolysis probes. Hydrolysis probes, an overview Hydrolysis probes are a popular detection chemistry for monitoring sequence-specific amplification in RTPCR. Just like with¬† SYBR…

04 Sep 2009 PCR, qPCR and qRT-PCR

qPCR: Plexor and SYBR compared

In my last article I introduced you to the Plexor System. And from that we already know that while in reactions that user SYBR Green for detection, fluorescence increases with accumulation of PCR product, with Plexor the fluoresence decreases. Today I want to compare some other well-known features of SYBR Green chemistry and see how…

24 Aug 2009 PCR, qPCR and qRT-PCR

Get the qPCR Fluorescence Low Down with Plexor

In real-time PCR, there are two primary ways to detect amplicons using fluorescent monitoring. One is intercalator-based dyes such as SYBR Green, and the other is probe-based techniques (hydrolysis or hybridization probes). All of these methods share a similar mechanism of measuring increasing fluorescence during amplification. But there is another completely different way to quantitatively…

20 Aug 2009 PCR, qPCR and qRT-PCR

Controls and Tips for TA cloning

Controls are obviously extremely important when setting up experiments. Without them, meaningful interpretation of the experimental results can be impossible. I say obviously, but in my previous job as a technical services scientist, you’d be surprised at how often I found myself talking to customers about the importance of controls. One customer commented, during our…

23 Jun 2009 DNA / RNA Manipulation and Analysis

DNA Jelly for Cell-Free Protein Synthesis

I had previously talked about the basics of cell-free protein synthesis and ways to improve yields from the technique. But recently I came across an article describing a technology that promises to dramatically improve the efficiency of cell free protein synthesis. The article, published in Nature Materials, shows a novel way to produce proteins cell-free…

28 May 2009 Protein Expression & Analysis

Solved:Low Yields in Cell-Free protein Synthesis

In my last article, I introduced Cell-Free Protein Synthesis. Today I want to talk about a major bottleneck in in vitro cell-free protein expression; low yield. Most often, paying attention to the important factors such as template purity & design, transcription and translation inhibitors and potassium and magnesium concentration will solve any problems with low…

21 Apr 2009 Protein Expression & Analysis

An Intro to Cell-free Protein synthesis

Cell-free protein synthesis (aka In vitro translation) refers to protein production in vitro using lysates generated that provide the cellular machinery necessary for synthesis. The lysates can be of bacterial or eukaryotic origin. It is a useful alternative to in vivo synthesis for generating protein for the study of things like: protein:protein interactions (pull-down assays,…

02 Mar 2009 Protein Expression & Analysis

Quantitative RT-PCR: One-step or Two-step RT?

qRT-PCR (quantitative reverse transcription-polymerase chain reaction) is now the gold standard technique for mRNA detection and quantification, sensitive enough to enable quantification of RNA from a single cell. The reverse transcription (RT) step is the main source of variability in a qRT-PCR experiment, so an optimal reverse transcription is essential for a reliable and successful…

16 Feb 2009 PCR, qPCR and qRT-PCR

Tips for Eukaryotic Cell Transfection

Transfection of eukaryotic cells is a routine but sometimes tricky procedure. There are several transfection reagents available on the market, but sometimes the old methods are the best. I find that the simplest, fastest and cheapest transfection method for eukaryotic cells is calcium phosphate mediated transfection (1). It’s main advantage is that, since Ca2+ is…

29 Jan 2009 Cells and Model Organisms