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Primer design can sometimes feel like more of an art than a science, and designing the best primer can significantly affect the success or failure of your experiments. Here are a few tips on optimizing primer design for several different applications: PCR amplification/cloning One of the most common primer-based applications is cloning. The desired amplicon…
The focus of my grad studies and postdoctoral research has been the analysis of proteins regulated by reversible protein phosphorylation. However, the number of unique facets in which protein phosphorylation can be studied is astounding, and is diverse as any niche of the biosciences. This article is the first in a series that will give…
What is HRM Analysis? It is now more than a decade since the introduction of melting analysis to characterize PCR products. Melting analysis following SYBR green-based real-time PCR has become a mainstay in research laboratories worldwide for applications such as gene expression because of it’s ease of design and cost-effectiveness (i.e. no need for expensive labeled probes)….
We recently featured an article about how to streamline your cloning. But what about those days when you have too much on your plate, and need to put some things off until later? Here are a few hints on where you can pause in your cloning experiments while working on other projects: Restriction digests can…
Northern and Southern blotting are now a thing of the past. They’ve been replaced with a faster and more quantitative technique. No longer do we wait days to know whether a gene is expressed. We can have the answer in 45 minutes! Real-time PCR is now commonly employed in almost all molecular biology laboratories to…
One of the very first things you need to do when getting set up for quantitative PCR (qPCR) is to determine the efficiency of the assay because knowing the assay efficiency is critical to accurate data interpretation. Here’s how.
I always keep an ear open for helpful tips in the lab – those little tricks that can make your experiments faster, easier and better. Here are a few tricks I’ve picked up for trimming down the time it takes to do your cloning: Restriction digests Many digests are complete within 10 minutes of digestion…
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…
If you have ever attempted to load a SDS-PAGE gel only to miss the well, stab the divider, and then watch helplessly as your sample squirts off into the wrong well, then this tip is for you. The fortunate among us are able to use pre-cast gels with the wells outlined on the gel plate,…
We received the following question from Bitesize Bio reader, Beheroze Sattha. It relates to a problem with absolute quantification using plasmids for standard curves. Since many people use this technique it is an interesting one question for us to explore, and it also gives us a great opportunity to cover some important tips for performing…
If you’re performing DNA/RNA precipitations, you will have read Suzanne’s excellent article on which alcohol to use for precipitating your precious samples (check out some useful info in the comments for that article as well). Its publication prompted the recall of a useful tip I learned from a post-doc many years ago, one of those…
There’s something in the water, and it would love to go after your experiments. Straight out of the tap, water contains microorganisms, endotoxins, DNase and RNase, salts and other impurities that could gobble up your experiment in one bite. Of course we avoid this drama completely by using purified water from which these nasties have…
Working at a plasmid repository, I get a lot of feedback from scientists who are relieved we exist simply because that means they don’t have to request a plasmid directly from another academic lab. Either they’ve had a bad experience making requests in the past, or they really don’t know how to go about doing…
TAE or TBE, which is best? Well, of course, it depends on what you want to do. Here are the pros and cons of both: But you might be better of using neither of these buffers. Despite the fact that they have been firmly established as the most popular buffers for DNA electrophoresis since their…
One of the great features of PCR is its excellent sensitivity as we know. And many articles describe real-time qPCR as an added leap forward in that sensitivity – to the point where it has become a standard feature of a new assay description Indeed, I’m currently developing a new qPCR assay to replace a…
If you’re like many researchers, problems with PCR amplifying high GC DNA templates will be a major annoyance for you. Many strategies developed to overcome this issue. Betaine is the most common PCR additive used to enhance amplification of GC rich sequences because of its ability to dissolve secondary structure that blocks polymerase action. But…
qPCR is a technique used daily in most labs, but the first step, designing your qPCR primers, can be the biggest obstacle to even getting started. Without a good pair of primers, you can’t start asking the real questions and generating data. And sometimes the effort involved in optimizing an assay for high efficiency and sensitivity…
It’s the molecular biologist’s version of ‘I have good news. . and bad news’. The good news is that I amplified the DNA band of interest. The bad news is that I amplified these other bands as well! Oh, and this smear. What to do? Typically you might try and cut out the band of…
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…
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…
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…
So you have some blood stored in the -20 °C or -80 °C and you want to isolate RNA from the samples. If you wanted DNA, you would have many products to choose from. But for RNA, your choices are more limited. Obtaining RNA From Frozen Blood is Difficult Why is that? The reason is that most…
The following question was emailed to Bitesize Bio by Beheroze Sattha and I gladly took up the challenge, and I immediately knew the answer. Or so I thought. After delving extensively into Pubmed, Genes V (I know, I need a new version) and Molecular Cloning I have come up with an answer, but it is…
Thanks to Bitesize Bio reader, Muthu Arumugam for contacting us about some problems he has been having with restriction digestion and clean up of DNA. I have boiled his query down to four main questions that are pertinent for most molecular biologists, so I hope that Muthu and everyone else can learn something from my…
In response to my last article, The Taq behind PCR, one of our readers, Bonnie Barrilleaux, asked whether DNA could naturally survive at temperatures that would denature it. It also begged the question; how do proteins stay intact and functioning at these high (55°C and up) temperatures? It turns out, cells do a lot of…
Gel extraction — what could be easier? Now we have quick and easy gel extraction kits, we no longer need to use time-consuming old fashioned methods like electro-elution or “freeze and squeeze”. Thank goodness. When Gel Extraction Goes Wrong But even the simplest of procedures can go wrong. Maybe you were distracted, confused, or thought…
Controls are obviously extremely important when setting up experiments. Without them, a meaningful interpretation of the experimental results would 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…
The reverse transcription (RT) step of RT-PCR for converting RNA to cDNA is critical for accuracy in quantification and for finding low copy messages. Thus, you want to make sure that this step is performed with the highest efficiency but without having to optimize every single step. To help you further in optimizing the RT…
Nobel Laureate Kary Mullis is generally credited with inventing the polymerase chain reaction, but his discovery owes a lot to a microbiologist who loved to travel, some refuted assumptions of what can live in hot springs, and a now-closed field station in Yellowstone National Park.Here’s the story. In the 1960s, Thomas Brock was a biologist…
Removing unwanted DNA from vectors is one of the most routine laboratory techniques in cloning. Check out our top tips.
No matter how many times you look at it, it’s not going to change. You are planning your next cloning experiment, but there’s a problem. The only restriction enzyme that cuts in a suitable position on your plasmid vector also, as luck would have it, cuts in another position elsewhere in the vector so you…
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 and design, transcription and translation inhibitors, and potassium and magnesium concentration will solve any problems with low…
After ligation, the method you use for desalting your sample prior to electroporation is critical, especially if your ligation is inefficient, according to a study by Schlaak et al [1]. Under standard electroporation conditions, the electric field of 12-18 kV/cm generated in a 0.1mm-gap electroporation cuvette means that the conductivity of the sample must be…
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,…
There is nothing more frustrating than getting back rubbish data from a DNA sequencing run, especially when you are waiting for an important result. For example, confirmation of that clone you have been trying to get for the past three months! A lot of the time, the quality of sequencing data is within your control….
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…
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…
You are not alone. Everyone makes a hash of their protein gel sometimes but this resource can help you work out what went wrong, and feel better for seeing gels even worse than yours.
Your DNA samples are precious so take care of them! Here are 5 ways that DNA can be damaged, so now you now what to avoid in the future.
Ever heard of polymerising agarose gels? I haven’t. If you think you have, read this.
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