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...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...

...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...

... 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. ...

...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...

...chromosomes. These long range DNA interactions are often detected with chromosome conformation capture (3C) based... of the drawbacks of 3C methods, but could only detect chromatin interactions at one location in the genome...CAPTURE 2.0, an updated version of CAPTURE that detects chromatin interactions at hundreds of loci at once... The chromatin “picture” is then developed by detecting these chimeric DNA fragments by PCR or next-generation...Cross-links are reversed so that DNA fragments can be detected by PCR or deep sequencing. A drawback of the...of CAPTURE 2.0 CAPTURE 2.0’s increased rate of detecting chromatin interactions opens the door for answering...proof-of-concept experiment to show that CAPTURE 2.0 could detect multiple chromatin interactions at once. Chromatin...

... CRISPR-based cytosine base editing. Nature Biotechnology, 41(5), 663–672. https://doi.org/10.1038/s41587...activity and therapeutic application. Nature Biotechnology, 38(7), 892–900. https://doi.org/10.1038/s41587...concurrent adenine and cytosine editing. Nature Biotechnology, 38(7), 861–864. https://doi.org/10.1038/s41587...engineered Cas9-cytidine deaminase fusions. Nature Biotechnology, 35(4), 371–376. https://doi.org/10.1038/nbt...optimization and ancestral reconstruction. Nature Biotechnology, 36(9), 843–846. https://doi.org/10.1038/nbt...editors generated from TadA variants. Nature Biotechnology, 41(5), 686–697. https://doi.org/10.1038/s41587...editor with low off-target activity. Nature Biotechnology, 41(5), 673–685. https://doi.org/10.1038/s41587...

...the detection of RNA, dsDNA, and now ssDNA. By comparing the ability of Cas12 and Cas14 to detect SNPs...adaptive immune systems that provide bacteria protection against infectious agents. Several enzymes behind...fantastic add-on to a diagnostic toolkit, called DETECTR, which has been created by the authors. This kit...kit uses Cas12 and Cas13 to quickly detect the presence of infectious organisms (ex: SARS-CoV-2) and genetic...SNPs using the DETECTR kit, the authors showed that improved specificity of Cas14 enables high-fidelity ...high-fidelity SNP genotyping. The development of  Cas14-DETECTR will soon enable the quick and efficient diagnoses...resources on the Addgene Blog Learn more about DETECTR Browse all of the CRISPR blog posts Download our...

...implications of using these technologies, but also the rapidity of the technologies’ adoption by the scientific...amongst the Nobel prize winners for developing the technology. Though these case studies are “old,” they lead...significantly over the last decade. He describes the detection of gravitational waves--a story I was much less...around the world. The paper describing the waves’ detection had over 1,000 authors. The chapter on deep learning...learning addresses the dilemma of creating a technology that could eventually replace scientists themselves...Addgene’s role in the rapid dissemination of these technologies, proposing that “Addgene, with its easy, fast...CRISPR-Cas plasmids, has been a key driver of the technology.” As an Addgene vet of 12 years, I’m very familiar...

... into plasmid DNA. In high throughput cloning techniques, although the specific sequences of the genetic...proteins and the availability of powerful imaging technologies, the combination of multiple fluorescent protein...-throughput cloning. MXS Chaining is one such technique and has been used to produce complex fluorescent...constructs. These fluorescent reporters can be used to detect structure and protein localization, as well as ...show robust labeling, with strong expression and detection of each fluorophore at the respective subcellular...that the separate modules combined using these techniques are free of specific restriction enzyme recognition...High Throughput Cloning Strategies Method Technology Pros Cons References Golden Gate Type II restriction...

... to) purification, detection, solubilization, localization, or protease protection. Thus far Plasmids ...proteins, which are sometimes used as tags for detection; however, those are just one (admittedly large...overexpress and purify proteins can face any number of technical challenges depending on their protein of interest...one-step purification with glutathione, can also protect against proteolysis. That’s one form of instability...it’s a good one), then your first step towards detecting, immunoprecipitating, or purifying that protein...Stability Good for purification with glutathione; protects against proteolysis, but may reduce solubililty... improves solubility; good for antibody-based detection SUMO ~100 amino acid protein  12 Stability At...

...thanks to these technologies but many questions remain. The use of other technologies such as optogenetics... cellular movements in the immune system. The techniques used here could be adapted to studying other ...labeled cells? 1. Epifluorescence microscopy This technique uses a fluorescence microscope where the light...excitation microscopy is a fluorescence imaging technique that allows imaging of living tissue up to about...from this volume. The consequence is that you can detect signal in thick specimens. See the video below ...monitor cellular movements in real time. This technique was first Introduced to immunology by three papers...papers in the journal Science in 2002. Now, this technique and the in vivo live-cell imaging (intravital ...

...not to say that the technique doesn’t have its limitations or that other technologies need to be discarded...relatively cheap (compared to many other technologies and techniques in the sciences). But how exactly does...the history of neuroscience research we have a technology that allows us to show causality in neural circuits...interest. Despite its power and versatility, the technique is remarkably straightforward to learn and relatively...behavioral optogenetics in order to implement the technique in my lab at The Rockefeller University. In this...exactly do you want to use optogenetics for? The technique is non-trivial; you don’t want to spend months...stereotaxic surgery is a neuroscience research technique often used beyond just optogenetics. Successful...

...University of Science and Technology in Shijiazhuang, defended the technology in a news article in Nature..., NgAgo. As you might expect with any nascent technology, researchers are having trouble getting NgAgo...early successes, and to find out what delivery techniques researchers are using. The detailed results of...Mary Gearing and discusses a newer, CRISPR-based technique to generate C->T or G->A transitions in a target...target sequence in live cells. This technique uses catalytically inactive CRISPR endonuclease (dCas9) fused...in this informative post. Interestingly, this technology may make some uses of Site Directed Mutagenesis...Software for Biology Adapting Toehold Switches to Detect Zika Virus As always, if you have any questions...

...avidin-biotin technology to improve cell adhesion on nanofibrous matrices.” Journal of Nanobiotechnology 13.1 ...in molecular biology for an array of different techniques and protocols. In this post we will discuss the...interaction with avidin for molecular biology and biotechnology purposes.  Once a polypeptide is biotinylated...reporter is then added to the reaction for easy detection. The multiple biotin binding sites found on avidin...amplification during these experiments. During detection, a biotinylated reporter enzyme that is pre-incubated...amplification of the reporter signal and easier detection. This strategy is referred to as the Avidin-Biotin...Zhu, Y. “A non-radioactive method for small RNA detection by northern blotting.” Rice 7.1 (2014): 1–7. PubMed...

...in the malaria community for the better (Nat. Biotechnol., 2014). Given the timing, it shouldn’t be a ...causes malaria). I attribute their success to technical expertise, thoughtful single guide RNA (sgRNA)...yet. As a genome editing tool, the CRISPR/Cas9 technology has been surrounded by a whirlwind of research...but generally rely on less powerful algorithms to detect off-targets. The few offline tools available mostly... sgRNAcas9. Top tools for transferring CRISPR technology to a new organism: For obvious reasons you can...developed with the purpose of applying CRISPR technologies to new organisms and will help you whether you...Report your findings to help everyone else. CRISPR technology has reached into so many different communities...

...translation, but a visual technique to study translation remained elusive. Two techniques developed by Addgene...mRNAs are digested with ribonucleases, ribosomes protect the segments of mRNA to which they are bound. These...translational frequency. Although highly useful, this technique requires extensive sample processing to avoid ...-mRNA complexes. Recent work has adapted this technique to monitor local translation and determine if ... single RFP-labeled osk and Oskar protein were detected at the posterior pole. Oskar protein levels were...compartment. Despite these limitations, these techniques represent powerful tools for studying translation...Protein Plasmids Check Out Other Hot Plasmid Technologies ...

...science and biotech due to its fermentation abilities and other useful traits. The “biotech yeast” You...You’re probably wondering if the “biotech yeast” is S. pombe or S. cerevisiae. Actually, it’s neither. .... The most commonly used yeast by biotech and pharmaceutical companies is K. phaffii (formerly known as... and advancements in other biomass production techniques in the following decades, Phillips made the decision...they abandoned the system. Since then, multiple biotech and pharmaceutical companies (along with some research...recombinant proteins by the pharmaceutical and biotech industries as a protein expression workhorse. This...Riley, R., et al., Comparative genomics of biotechnologically important yeasts. Proc. Natl. Acad. Sci.,...