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...Importantly, conventional flow cytometry uses one detector to detect one fluorophore and therefore one cellular...fluorophores can “spill over” into detectors that are not the primary detector for that fluorophore. For example...single (primary) detector to identify a fluorophore, spectral flow cytometers use all detectors for all fluorophores...panels: five laser spectral flow machines have 64 detectors, allowing for the use of up to 64 markers in a...emitted by a fluorophore is guided to a single detector using a series of mirrors and filters. Importantly... FITC signal is spilling over into the primary detector for PE. In order to correct for this, compensation...the fluorescent molecules can be quite broad, detectors will usually only pick up a small portion of that...

... and can be used in a multitude of scientific techniques such as molecular cloning or molecular diagnostics...used for an array of different molecular biology techniques and experiments. End point PCR’s goal is to amplify... cloning to create a plasmid of interest, for detecting the presence of an insert or piece of DNA in a...reaction (qPCR) also known as real time PCR is a PCR technique used for measuring a starting DNA concentration...during the PCR reaction cycles is continuously detected via a fluorescent signal. The signal increases...concentration. The cycle in which the unknown DNA is detected compared to the standard curve can be used to ...right. ddPCR uses a water-oil emulsion droplet technology that fractions the PCR reaction sample into approximately...

...Scientist at the Broad Institute, John Doench. CRISPR technology has made it easier than ever both to engineer...is true when designing gRNAs for using CRISPR technology – the “best” gRNA depends an awful lot on what...Gene knockout by NHEJ Gene knockout with CRISPR technology is usually accomplished by Cas9-mediated dsDNA... Created with BioRender.com. Two newer technologies offer an alternative to HDR for introducing ... another target C or A in the window. Another technology, prime editing (reviewed in Anzalone et al., ... although these are still early days for this technology, and the user may need to optimize numerous parameters...CRISPRa (activation) and CRISPRi (inhibition) technologies – a nuclease-dead Cas9 (dCas9) is directed near...

.... However, most SRM techniques also require specialized instruments and technical expertise, limiting ... model systems, paired with SRM techniques, and adapted for detecting nucleic acids as well as proteins...biological features previously only visible via techniques like super-resolution microscopy or electron ...Super-resolution microscopy (SRM), a family of imaging techniques developed to break through the resolution barrier...some of the limitations?  A “super-resolution” technique that uses common reagents, straightforward protocols...there is always a catch - some limitation of the technique that users should be aware of - and ExM is no ...microtubules. This issue is shared by other SRM techniques and can be alleviated by maximizing label density...

...learning a new technique while visiting a lab, or maybe even training on a new technique with a coworker...research group, you will also typically receive technical mentorship from a ‘bench mentor’. In some instances...mentor is a staff scientist, postdoc, lab manager, technician, or a graduate student. This is the person you...basis who will help you get on your feet with techniques in the lab (and possibly much more, depending...difficult for you to become proficient in lab techniques, which will hinder your progress. You will also...serve two purposes – familiarize you with the techniques you will be learning and the biology you will...there for you to be independent performing the technique you trained to do? Walk through the protocol mentally...

...Förster) Resonance Energy Transfer, technique which allows the detection of molecules' interactions, modifications...fluorophores to proteins, pioneers of this technique were able to directly detect protein/protein interactions in...dissociations in situ. Used since the mid-90s, this technique has revolutionised the way we apprehend molecular...playing “live.” Indeed, FRET was one of the first techniques which enabled the measurement of single molecule...microscope. Historically, molecular interactions were detected by indirect means often using probes with the ... fluorophore can be measured using microscopy techniques. FRET measurement sensitivity makes it suitable...DNA and proteins. FRET, in theory, is an easy technique, but it is very important to follow some simple...

...to get right. If you’ve thoughtfully done the technical design of your blot, but are still finding yourself...Most people will simply load an extra ladder or technical replicate (ie, leftover sample) to fill the relevant...you may want to consult Tables 1 and 2 in our Technical Design of a Western Blot post to make sure you...transfer. Troubleshooting The two most common (non-technical) issues with transferring are smaller proteins...than just high background, your issue is likely technical. White circles, for example, could indicate that...incubation steps.  Many errors can arise from technical issues. The good news is that such errors are ...means the amount of protein loaded is outside the detectable range of that antibody dilution. Adjust the antibody...

...chromosome, which was technically challenging. Other homology based technologies We've described Sequence...alternatives to restriction enzyme cloning. These newer technologies have become more and more common, and for good...Overview of Gibson assembly The Gibson assembly technique was first described by Dr. Daniel Gibson and colleagues... 5 days. One drawback to the Gibson assembly technique is that the process works best with fragments ...Gibson assembly combined with the popular CRISPR technology and is described in a recent publication by the...When followed by the standard Gibson assembly techniques described above, this resulted in the direct ...Cloning (System Biosciences) In-fusion Cloning (Clontech) Although these kits may come at a high price...

...Fluorescent imaging techniques have become indispensable tools for molecular and cell biologists over... Osaka University have developed Nano-lantern technology. Nano-lanterns contain a Renilla luciferase variant...but it has precluded widespread adoption of the technology. In 2012, Saito et al. described the first Nano-lantern...luciferase (RLuc8) with Venus. This luminescent technology is based on the principle of bioluminescence ...to excite the fused fluorescent protein. This technique is very similar to the widely used FRET, but it...ATP sensors based on Nano-lantern, showing the technique’s versatility and adaptability to many different...optogenetic system. Biosensors based on Nano-lantern technology are also very robust, with an eNL-based calcium...

...please read on to learn more about this exciting technology from the folks at Transposagen. This post was...engineering approaches. Most importantly for this technology, however, is that the transposase also enables... with no known upper limit [1]. The piggyBac™ technology can be used for numerous applications, including...of interest. Fortunately, by combing nuclease technology with the piggyBac™ system, these challenges can... DNA using the piggyBac™ transposon selection technology using a site-specific nuclease to stimulate homologous...of using piggyBac™, you could use recombinase technology (Cre/loxP) to remove the donor cassette. While...summary, the piggyBac™ transposon is a powerful technology that may be used for a variety of research applications...

...things more interesting, we are working in a new technical discipline that nobody understands (synthetic ... However, since the lab was highly focused on protecting intellectual property, it worked away in isolation...ideas (how to stop rhino horn poaching using biotechnology) were thrown on the table and dissected with...that survived the literature review, survived technical feasibility analysis, and was a marketable product... our day job, we worked on plants engineered to detect explosives and rapidly indicate their presence....reputation. The public is can be fearful of this technology and angry at the agriculture companies that produce...GMO food plants. When a press piece ran on the detector plants, we expected to see that same anger. Instead...

...Spectrophotometers. NEB Tech Note (2019).  Loughrey S and Matlock B. Detection of Protein in Nucleic Acid...Thermo Fisher Scientific Tech Note (2016).  Loughrey S and Matlock B. Detection of phenol in nucleic acid...Fisher Scientific Tech Note (2017).  Extinction Coefficients. Thermo Fisher Scientific Tech Tip (2013).  Additional...characteristic absorbance spectra, which you can detect by examining the A260/280 and A260/230 ratios.  ...potential contaminants, some of which are easier to detect by spectrophotometry than others. The ones most... to other reagents, making it more difficult to detect. A particularly strong absorber at 230 nm is guanidine...However, such systems are no substitute for sound techniques, routine machine maintenance, and critical interpretation...

...enhanced detection of the product. As an added benefit, replicates are built into the technology. One well...applications for the technology.  Droplet digital PCR is well suited for the detection of low copy numbers... the detection of rare sequences and single cell analysis. It is also being used in the detection of microbes...approximately 20,000 droplets using water-oil emulsion technology. Each droplet contains the ingredients for amplification... Most droplet readers have a few channels for detecting fluorescence so that it is possible to measure...applications include measuring viable probiotics and detecting circulating pathogens.  With its many applications...Central PMCID: PMC3991984. Song, Neng, et al. "Detection of circulating Mycobacterium tuberculosis-specific...

...molecules is necessary for a detectable signal, limiting the sensitivity of the technique. One potential solution...It seems that there’s a new CRISPR advance or technique published every week! One of the newest applications...make this system truly advantageous. This new technique, developed in Thoru Pederson’s lab, brings us ...to mapping pairs of intrachromosomal loci. The technique successfully resolved loci with physical map distances...modifications and applications Ma et al. developed this technique using standard fluorescence microscopy, and are...extending the potential imaging time for this technique. The NIH Common Fund has made mapping the 4D nucleome...of genomic loci. Ma et al. anticipate that the technique will be applicable to studies of cell cycle progression...

...the capabilities of fluorescence-based techniques in biotechnology. Recent examples of advancements include...These dyes can also be attached to an antibody to detect various targets, such as proteins, nucleic acids..., which are pivotal in their application in biotechnology (Grimm & Lavis, 2022). These variations directly...and acceptors in their product descriptions, or technical manuals. Specific applications and matching dye...several factors. Firstly, brighter dyes enable detection of finer details by improving signal-to-noise ...designed for use in super-resolution microscopy techniques, such as Stimulated Emission Depletion (STED)...-fluorescent light when excited. While it is technically cell-permeable, its use in live cells can sometimes...

...correlated to behavior, the technique is relatively slow and is not suited to detect rapid changes in DA concentration...slices Demonstrate DA detection in a single neuron in vivo in Drosophila Detect DA dynamics in response...extracellular fluid (Darvesh et al., 2011). This technique is performed with a probe surrounded by a semipermeable... lab has developed dLight1, a molecular tool to detect and image DA in live animals (Patriarchi et al.... kinetics (10 ms on and 100 ms off), enabling detection of individual peaks of DA release.  Using dLight1... sensors are powerful new developments in the detection and measurement of neurotransmitters in various...fluorescent sensor enables rapid and specific detection of dopamine in flies, fish, and mice." Cell 174.2...

...found on immune cells. However, there are more techniques and strategies one can employ beyond simply incubating...downstream application, one or several of those techniques can be used alongside standard surface labels...introduce you to a few useful labeling and staining techniques beyond standard surface labeling. Pro tip! Many...and 'staining' interchangeably to refer to any technique that combines a marker with a signal. For clarity...labeling While antibodies used for target marker detection are usually conjugated to a fluorophore, not every...cases, a good alternative is using a two-protein detection system, as is commonly used in western blotting...proximity to the target marker, allowing for its detection. The advantage of this system is that streptavidin...

...especially in in vivo models, and new Cre-lox based technologies are still being developed today. In this post...differences amongst the Brainbow systems. Pre-Brainbow techniques, such as Golgi staining, injection of diffusible...time-intensive, necessitating the development of a new technique. In a structure as tightly packed and interwoven...name Brainbow is truly fitting for this colorful technology. Optimizing the system: Brainbow-3 Although Brainbow... et al. released a refinement of the Brainbow technology, Brainbow-3.0, with the goal of overcoming the...mutant YFP that does not fluoresce, but can be detected via immunostaining. This feature facilitates screening...Karine Loulier, et al. (2014). Currently, Brainbow techniques are also being applied to study model organisms...

...simple technology,” Doudna further explained. “That’s why we’re seeing adoption of the technology in different...University.  There can be no doubt that CRISPR/Cas9 technology has been a breakthrough for the genome-editing...CRISPR for complex genetic diseases As CRISPR technology becomes increasingly more cost effective and ...CRISPR/Cas9 in comparison to other genome editing techniques is its ease of use.  “I was really excited when...of those off-target mutations when using this technology. When CRISPR therapies are being given in vivo... “A glance at genome editing with CRISPR-Cas9 technology.” Current genetics. https://doi.org/10.1007/s00294... Khatibi et al. 2021.  “CRISPR Genome Editing Technology and its Application in Genetic Diseases: A Review...

... most cell types. But prime editing, a CRISPR technique developed in David Liu’s lab in 2019, has added...Nature, is a “search-and-replace” genome editing technique that mediates targeted insertions, deletions, ...Liu lab contributed an improved prime editing “architecture” that can be combined with any of the PE2-PE5...with improved stability Traditional sgRNAs are protected from cellular degradation by Cas9 binding, but...target site, but it cannot drive prime editing. To protect the 3′ end, the Liu lab appended RNA pseudoknots...mammalian cells: the small RNA-binding exonuclease protection factor La. They found that La, which is ubiquitously...DNA sequences with twin prime editing. Nature Biotechnology, 40(5), 731–740. https://doi.org/10.1038/s41587...