...consists of prominent members of the bioscience and tech transfer communities who help guide and promote ...that had Platforms with a capital ‘P’ focused on technology ... has been critical to the Broad’s success ...biological questions, you need a tremendous amount of technology development alongside it.” Doench starting working...Perturbation Platform and encompasses a variety of technologies to functionally investigate the human genome...genome. Doench says he enjoys working on technology development under the Platform model at the Broad Institute...Institute because they are “not just developing technology for its own sake,” but work in true collaboration...history of RNAi left him a bit skeptical of the technology at the start of the CRISPR craze, but he says...

...microscopy technique for your study see Addgene’s blog post Which Fluorescence Microscopy Techniques is Best...fluorochrome is removed from all other detectors except its dedicated detector. To ensure proper compensation ...cell health, analyzing cell cycle stages, and detecting specific populations of cells in a complex tissue...sample (1). When combined with cell sorters, this technology can be used to isolate a specific subset of cells...fluorescence intensity.   What flow cytometers detect Cell size and complexity, measured using visible...directions. The degree of scatter is captured by detectors and provides useful information about the cell...emits a fluorescent signal that is captured by detectors. The strength of the emitted photons varies from...

...CRISPR 101" post. Next-generation sequencing technologies are the gold standard but they remain cost-prohibitive... assays (e.g. Surveyor® or T7E1) for mutation detection. While these methods paved the way for mutation...introduce digital PCR as an emerging validation technology. Digital PCR has several advantages over mismatch... require standard curves, and can theoretically detect a single target molecule in a sample. Digital ... real time PCR. These assays can be designed to detect either a donor sequence integrated through homology-directed... remainder of this post will focus on assays to detect NHEJ. These assays include a forward and reverse...between 200 and 500 ng of purified PCR product. Low detection limits: Digital PCR assays can accurately quantify...

...popular technique in neuroscience and developmental biology. This type of cell labeling technique involves...group developed the original iteration of this technology in 2007. Used to label neurons in the cerebellum...cerebellum of mice, the team appropriately named their technology Brainbow. Brainbow 1.0 used the fluorophores ...construct but do not undergo recombination can be detected by immunostaining of the truncated YFP. In Thy1...CMV-Brainbow construct at the embryonic stage. This technique could be used to target Brainbow to neurons by... promoter. Zebrabow is an extremely versatile technology as recombination can be induced in developing...cells. Tetbow optimizes Brainbow for current technologies Tetbow (Sakaguchi et al., 2018) overcame the...

...Einstein College of Medicine has recently published techniques to follow translation in living single cells....cells. However, before CRISPR-Cas9 these techniques were quite tedious and, even here, there is the question... symposium seeking inspiration to resolve the technical hurtle of monitoring protein turnover of specific...interesting talk by Prof. Erin M Schuman, a former Caltech researcher, now Director at the Max Planck Institute...Frankfurt am Main, Germany. She presented a novel technology, back then still unpublished, that allowed the...synthesized proteins with FUNCAT Professor Schuman’s technique applies metabolic labeling using a noncanonical...noncanonical amino acid, azidohomoalanine (AHA). In the technique, AHA is incorporated into newly synthesized proteins...

...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...

... permeabilization techniques — and so it may be necessary to try multiple techniques. For example, we... ability to detect endogenous levels of the target protein. It may only be able to detect the target protein... (or refute) that the antibody is selectively detecting the target-of-interest in your assay and sample-of-interest...must first confirm that a candidate antibody can detect the antigen of interest in the given protocol. ...supportive, the data must show that the antigen being detected in the intended application — here, flow cytometry...conclusively prove the ability of an antibody to detect the antigen of interest when expressed at endogenous... approach, you’re looking for an antibody that detects the target in the parental cell line, but not in...

... 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...

...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...

...the mice, these “no needles” techniques may come in handy. These techniques are referred to as “instillation...to conducting mouse experiments. Some of these techniques are what we call “beginner”, or injections that... practice after initial mouse training. Other techniques require more skill and additional training, going...Image created with biorender.com.   Needle techniques Intravenous Intravenous (IV) injections are most...easier of the two, but it actually requires more technical skill to place the needle in the correct vein ...are probably the most advanced needle injection technique. Intrathecal injections are used when the substance... ml (10 µl) Skill required: expert No-needle techniques The term “injection” is most often associated...

.... 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...

...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...

...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...

...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...

...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...

...Fluorophores Fluorophores allow you to detect your antibodies using techniques like fluorescence microscopy, flow...conjugate various labels to antibodies that produce detectable signals like light or color. In this post, we...substrates for each of these enzymes allow you to detect enzyme activity (and therefore your antibody) either... light produced by a chemical reaction, can be detected using specialized imaging instruments or X-ray...biotin conjugated antibodies good options for detecting low abundance targets. Check out our post on Biotinylation...mention. Oligonucleotides allow for sensitive detection of low abundance targets with significant multiplexing...JL, et al (2020) Proximity Ligation Assay for Detecting Protein-Protein Interactions and Protein Modifications...

...Education Project (pgEd). Advances in genetic technologies and other biomedical innovations promise an ...society are to figure out the best way to shepherd technologies through thorny ethical issues, ensure that everyone... fruits of scientific research, and prevent technological advances from exacerbating existing inequalities...about genetics, both as individual consumers of technologies and as societal participants in the political...public health, science policy, and science and technology studies (STS). I also worked with a number of...field of interest. We often communicate in very technical and jargon-filled language in academic writing...out opportunities to communicate science to non-technical audiences. Many universities or cities have organizations...

...collection page. SARS-CoV-2 detection tools There have been many detection tools developed over the last...methods to use CRISPR to detect SARS-CoV-2. Other protocols and tools for detection have been made openly...Zheng’s lab had already developed nucleic acid detections tools based on Cas12a and Cas13  which could ...could be repurposed for SARS-CoV-2 RNA detection. Early in the pandemic, labs around the world have been modifying... modifying these tools for COVID-19 detection with the ultimate goal to develop low cost and quick point-of-care...that can be used in a one-step RT-qPCR reaction to detect SARS-CoV-2 nucleic acids. Mammalian targets During...2020) Open Sharing During COVID-19: CRISPR-Based Detection Tools. The CRISPR Journal 3:142–145. https://doi.org...

... can be a lot of work — and what if you want to detect more than one protein in your sample? Or what if...! Rather than start over, you can remove the detection antibodies from your membrane, allowing you to...stringent stripping methods. If you are planning to detect multiple proteins through several rounds of stripping...the strength of bands from different rounds of detection. Finally, stripping and reprobing works best with...blots that use chemiluminescent or fluorescent detection, like horseradish peroxidase (HRP) or Alexa Fluor...Fluor dyes, respectively. Chromogenic detection produces colored precipitates that permanently stain the...it. Ideally, dry your membrane once before any detection steps to avoid retaining residual antibodies. ...