... is toxic. When handling, use proper personal protective equipment including laboratory coat, laboratory...

...meter Stir plate Magnetic stir bar Sonicator Ear protection Vortex Reagents Adherent HEK293T cells (ideally...

...to lysis by freeze-thaw if not suspended in cryoprotectant like Glycerol or DMSO solutions. Quantify the...

...They also have a limit of detection of ~5%. This means they do not reliably detect editing events that occur...primary methods of off-target detection: biased and unbiased. Biased techniques only sequence certain sites...: Biased Genotyping Options Technique Sequence Information Detection Limit Advantages Disadvantages...highly sensitive with detection levels as low as .01% (Hendel et al. 2015). Low detection rates mean the investigator...off-target mutations if they remain undetected using these techniques. Frequencies of off-target mutations...have a minimum detection limit of 0.1% (Tsai et al. 2015). This contrasts with detection frequencies of... Table 2: Unbiased Genotyping Options Technique Detection Limit Application Advantages Disadvantages...

... for detection RANbody is detected enzymatically with HRP substrate More sensitive than detecting antigen...require a secondary antibody for detection. Instead they are detected with tyramide signal amplification...and is useful for detecting GFP. Spaghetti Monster tagged RANbodies can be detected either with a labeled...sensitive than HRP RANbody detection, it is approximately as sensitive as detecting the same antigen with ...RANbodies are easily detected with a labeled anti-chicken antibody. Chicken IgY RANbody detection is less sensitive...sensitive than HRP RANbody detection but approximately as sensitive as antigen detection with a standard primary... When detected with a labeled anti-HA or -MYC antibody, approximately as sensitive as detecting antigen...

...different biotech companies which are enzyme-based mycoplasma detection kits. These kits detect mycoplasma...irreplaceable or highly expensive). Detecting mycoplasma contamination Detection of mycoplasma by naked eyes ...Another method used for mycoplasma detection is using PCR. In this technique, PCR primers specific to the 16s...cells that can detect the presence of mycoplasmas in the culture. Once these cells detect mycoplasmas, ...Assistant at MGMs Institute of Biosciences and Technology. If you are reading this article then you probably...through cross-contamination and due to poor lab techniques. These include reusing pipettes for multiple ...turbid nature, contamination is difficult to be detected in the serum that is most commonly used for every...

...that require scientists to detect proteins in their samples. One technique that relies heavily on antibodies...Enzyme-linked Immunosorbent Assay. ELISA are used to detect proteins within a 96- or 384-well, flat-bottomed...bottom of the well), and then its concentration is detected through visualization of an enzymatic reaction...ELISAs, which differ in the way the antigen is detected. These are sandwich, competitive, direct, and ...available ELISA kits are for a sandwich ELISA. This technique works by first coating the wells of a flat bottomed...specific to the protein you are interested in detecting in your sample. When an ELISA kit is ordered, ... your protein of interest.  The next step is to detect the protein-antibody pair. In a sandwich ELISA,...

...Addgene.org Addgene Help Center: Tech Transfer Technology Transfer Information Tech Transfer News: A Reintroduction...get an Addgene account for Tech Transfer Offices. They’re packed with tech transfer resources (summaries... question? Refer them to Addgene’s Tech Transfer Team: techtransfer@addgene.org Congratulations to the...stats and strategies with tech transfer offices. Drop us a line at techtransfer@addgene.org. Albert Einstein...’re trying to make MTA turnaround faster with technology transfer offices (TTOs). The latest of these ...deliver the very first ones in person by our own Tech Transfer Team! Why MTAs Matter Though they may seem... important—they guarantee certain rights and protections for depositor and requestor alike. That’s why...

...retroviral enChIP system using CRISPR technology. Can you describe this technology and some of its potential applications...development of new biological technologies. The locus-specific ChIP technologies consisting of iChIP and enChIP... to develop new technologies. Majority of them are eager to just "import" technologies developed abroad...The Fujii lab specializes in developing novel technologies to analyze molecular mechanisms of genome functions...extensively with enChIP systems. Can you describe this technology and its advantages? Fujii: In the last several...years, my lab has been working on development of technologies for biochemical analysis of genome functions... chromatin immunoprecipitation (ChIP) (iChIP) technology, which uses locus-tagging and affinity purification...

...cannot imagine any use for GM technology other than to sustain those techniques. We feel strongly that the...transformation technologies like those pioneered by Ewen Mullins of Ireland’s Teagasc.  Plant biotechnology is full...there are a lot of unexplored areas in plant biotechnology that are ripe with opportunity. We’ve decided...One of the biggest difficulties that plant biotechnologists face is public perception of genetic modification... crops has burdened this emerging engineering technology. Monoculture and chemical spraying, mainstays... current benefits and future potential of the technology to the public in an open, responsible manner....this challenge directly by developing a plant biotechnology product specifically for consumer sales. Flowers...

...genetically encoded photocaging technique to insert a light-removable protecting group, specifically a nitrobenzyl...have been making major improvements to CRISPR technology since its initial applications for genome engineering...prime editing, anti-CRISPR proteins, and other techniques all aim to improve targeting specificity or reduce...2015, scientists combined CRISPR and optogenetic techniques to develop a variety of photoactivatable CRISPR...spatially control gene expression. Both of these techniques take advantage of light-induced dimerization ... their CRISPR system. This common optogenetic technique can be employed in other split systems where light...activated with light and lowered background DSBs (non detectable). Like their prior work, the Sato lab also showed...

...LDR can reliably detect single-base differences. The original creators of this technique visualized the ...Development of a ligase detection reaction/CGE method using a LIF dual‐channel detection system for direct ...-based ligase detection reaction assay using a molecular beacon probe for the detection of low-abundance...other thermostable DNA ligases. These applications detect specific nucleotide sequences with high specificity... (SNPs) (5,12,13). For example, in the Ligase Detection Reaction (LDR), probes complementary to the sequence...methods to greatly increase the sensitivity of detection (14-20). LDR has also been extended to multiplexed...multiple potential SNP sites (15). The related LCR technique uses two sets of probes to achieve exponential...

...labeling strategy to detect cell-cell interactions in vivo (Ge et al., 2019). This technique relies on an S.... with biotin using avidin coated purification techniques. Due to the high affinity of biotin for avidin...proteins The first biotin based proximity labelling technique, BioID, was developed in 2012 in the Brian Burke... developed as well as new proximity labelling techniques. These newer tools are also currently available...combines a protein fragment complementation (PCA) technique with proximity labelling to validate binary protein-protein...proteins in one simple experiment. PCA is an in vivo technique where two proteins are fused to complementary ...Split-BioID blog post.  RaPID: RNA-protein interaction detection So far, we’ve covered ways to study protein-protein...

...emerging technology. We could do this for CRISPR/Cas9 – or any other germline editing technique—too. As... of Rogue Bioethics. On November 25, the MIT Technology Review dropped a bombshell report. A scientist... is unethical to use scientifically unproven technologies to edit a human embryo, or have a woman carry...published on December 13 shows us, CRISPR/Cas9 techniques are continually being refined; it is completely...will eventually have available genome editing techniques that are sufficiently safe to manipulate an embryo—even... the June 2012 report on the ethics of “Novel techniques for the prevention of mitochondrial DNA disorders...disorders.” This document is about the popular technique incorrectly known as “three-parent babies,” where...

...mixture of cells. The technique of using fluorescently-labeled antibodies to detect proteins is referred...whole organs. Because the tissue architecture is preserved, this technique allows you to not only visualize...the same experiment because the reagents used to detect the presence of an antibody depends partially on...the primary antibody and provides the method of detection. You’ll also want to use the appropriate controls...applications. Western blot Western blots are used to detect the presence of proteins from samples containing... a secondary antibody allows the protein to be detected by chemiluminescence or fluorescence. Western ...immunosorbent assay (ELISA) Similarly, ELISA is used to detect the presence of a single protein from a heterogeneous...

...system, detect post-translational modifications, quantify protein levels relative to standards, detect the..., or western, is one of the simplest methods to detect the presence or absence of a protein (Renart et... size, (2) transferred to a membrane, and  (3) detected using antibodies.   Figure 1: Overview...finally visualized.   This process allows you to detect a single, specific protein within the complex mix...cells or tissues. Westerns are useful not only to detect the presence or absence of a protein, but can also...validated for immunoblotting. Different immunoassays detect proteins in different states. For example, in immunoprecipitation...protein in its native state while a western blot detects denatured polypeptide chains. The protein regions...

...enzymatic signal detection, users provide a substrate that the enzyme converts into a detectable product. Scientists... in a sample and then a second detection antibody binds. The detection antibody can be directly conjugated...then adds an antigen-specific detection antibody.  Importantly, the detection antibody and the capture antibody... either directly conjugated detection antibodies or unconjugated detection antibodies followed by conjugated... specificity, and sensitivity (a good ELISA can detect picogram quantities of the target!) and use them...conjugated with a reporter or tag that can then be detected.  Typically, the reporter is an enzyme such as...abundance antigens may want to consider alternative detection methods.      Figure 1: The four types...

...enrichment and structure determination LEA proteins protect samples during cryo-EM grid freezing Engineered...these limitations, the Funabiki Lab developed a technique called Magnetic Isolation and Concentration (MagIC...and in complex with its chaperone NPM2, but the technique should be widely applicable to other targets. ...doi.org/10.1101/2024.01.21.576499.    LEA proteins protect fragile samples during cryo-EM grid plunge freezing...proteins that naturally adsorb to the AWI, can protect cryo-EM samples from damage (Abe & Lim, 2024).  ...complexes may dissociate  (right). B) Protein samples protected from AWI by helical LEAs. C-E) cryo-EM single-... Lim, 2024 under CC-BY-NC-ND 4.0 license. Protection by the LEAs allowed the authors to determine ...

...panel to detect live, transduced, and CD45-expressing cells. Marker Fluorophore detection Live... reporter, you can infer its presence through detecting the reporter's fluorescence. Another way to label...different markers of interest, you can separately detect these markers in your analysis. Lastly, there are... to the signal strength of the emitted photons detected by the cytometer. Brighter fluorophores have a...the signal-to-noise ratio, and the better the detection of the target marker. Looking at Table 1, we can...into each other, it means that the cytometer could detect a certain photon to be emitted by either of the...the two. As a result, inaccuracies in the detection of fluorophores (and the markers they label) can occur...

...effective method for detecting plasmid multimers. Digests themselves cannot be used to detect multimers, since...Electrophoresis (CGE) analysis Another technique that can detect multimers is capillary gel electrophoresis...upstream production. Biotechnol Adv. 2009 Jul-Aug;27(4):353-70. doi: 10.1016/j.biotechadv.2009.02.003. Epub... the plasmids, including long-read sequencing techniques, such as Oxford Nanopore sequencing or PacBio...via antibiotic selection (Crozat, 2014). How to Detect Plasmid Multimers Run an undigested plasmid on ...sequences within a plasmid, it cannot be used to detect plasmid multimers. Some companies provide NGS whole...SMRT sequencing. These longer reads are able to detect large repeat sequences within a plasmid and, therefore...

...activity mapping with STARR-seq   Although many technologies have been developed for the study of enhancer...and drug resistance. Recently, the Wu Lab at Texas Tech developed a pooled CRISPR knockout library and used...deep sequenced to identify the gRNAs that confer protection to WNV. From this screen the authors were able...assembly utilizes 5 different overlap sites, this technique also allows for easy and rapid targeting of complex... and mutant library generation in several biotechnologically and clinically relevant bacterial species...SnoopCatcher The development of recombinant DNA technology has allowed molecular biologists to fuse fragments...through Addgene. Stovicek, et al. J Ind Microbiol Biotechnol. 2015. PubMed PMID: 26376869 Jensen, et al. FEMS...

...received a Ph.D. in Biology and Biotechnology from Worcester Polytechnic Institute. What is RNAi? The loss-of-function...earned his Ph.D. in Biology and Biotechnology from Worcester Polytechnic Institute where he investigated...the inherent advantages and limitations of any technique in the context of the biological question. For...unattractive from a practical perspective. Therefore, techniques that function downstream of DNA, such as RNA ...interfering RNAs.   RNAi vs. CRISPR: Complementary techniques It is fair to say that CRISPR has revolutionized...With the meteoric rise and diversity of CRISPR technologies, one might incorrectly relegate RNAi to history...requires the presence of dsRNA. In plants, CRISPR technologies are predominantly used to disrupt function at...

...very specifically to target molecules. Using a technique called systematic evolution of ligands by exponential... used in a variety of applications, including detection of environmental pollutants, drug discovery, and...a “bad batch”); Customizable sensitivity and detection - since they frequently undergo conformational...oligonucleotides in fluorescent visualization techniques for decades, most notably for fluorescent in ...visualize RNA. Low signal-to-noise ratios with such techniques can be problematic, however. Well designed aptamers..., thereby improving traditional visualization techniques. In addition, their incredible versatility opens...Fluorescence resonance energy transfer (FRET) technology has been studied and used in assays for over ...

...disease earlier. New techniques are currently being tested in clinical trials to detect mesothelioma earlier...diagnosis, the hope is that as these new detection techniques and therapies emerge into mainstream use... asbestos. However, as the U.S. Environmental Protection Agency has noted, there is no safe level of asbestos...growth of tumors. More often than not, multiple techniques are used as part of a treatment plan, an approach...intraperitoneal chemotherapy (HIPEC). With this technique, surgery is performed to remove the mesothelioma...delivered intravenously. New treatment options: Early detection, immunotherapy, and angiogenesis inhibitors New...life expectancy of mesothelioma patients: early detection and immunotherapy.  It has long been known that...

...people find it can detect even smaller differences, and it can even be used to detect a single copy of ...which aims to increase your understanding and technical proficiency with common applications - now let... thumb, you can assume that qPCR can accurately detect expression differences of twofold or greater with...Can be multiplexed Single amplicon validation technique Hydrolysis Probes (Taqman) Low; can be reduced...Biassoni, 2014). At the bench As a highly sensitive technique, qPCR needs a steady pair of hands to produce ...of triplicates to understand variation due to technical error.  Plate readouts can be affected by plate...differences may be a concern in your reaction, analysis techniques that use raw data corrections are a better choice...

...proteins may not be detected at all, while overloaded proteins may saturate the detector. In between these...extremes lies the linear detection range. Figure 4: The linear detection range of the assay is between...loading control points are outside of the linear detection range. As a result, they have almost the same ...computational quantification. Outside the linear detection range, band intensity measurements become extremely...are often called “housekeeping proteins” and are detected with an antibodies, just like the POI. This approach...means that at sample concentrations appropriate to detect a less-abundant POI, the loading control will almost...error, it’s important to determine the linear detection range of all proteins that will be measured in...

...contaminants and to subsequently develop technologies to protect fresh water repositories in Mexico. At ...insecticidal proteins to the development of renewable biotechnology products and biosensors for herbicides as well...a bacterial expression system for renewable biotechnology products Dr. Idah Sithole-Niang, University ... Lecturer and a Plant Molecular Biology and Biotechnology expert. In order to preserve Cassava yields ...Robertson aims to make use of the CRISPR/Cas9 technology to engineer plants that are able to destroy DNA...Associate Professor (Right), focuses on molecular biotechnology for improving expression of nutritionally and...Bao Ton (center) is interested in applying gene technology to bacteria and plants. Her research involves...

... a revolution in genome engineering, with new techniques rapidly being developed. Performing a CRISPR ...potential for off-target effects. Cas9 RNPs are detectable at high levels shortly after transfection, and...plasmid based methods. One of the most common techniques for delivery of RNPs is electroporation (A in...the cytoplasm. In addition to the use of this technique in cell culture, it has also been applied to genome...combined with cell-type specific reagents in a technique known as nucleofection, which forms pores in the..., allowing for entry of a DNA template. Other techniques, such as lipid-mediated transfection (B in the...using this method for in vivo applications. This technique utilizes Cas9 proteins harboring receptor ligands...

... commercially available mycoplasma detection kits that use detector cell lines, ELISA, fluorometry, or...Incidence, sources, effects, detection, elimination, prevention.” Cytotechnology 39.2 (2002). 75-90. Pubmed... lines, or infected personnel and can persist undetected; unlike infections with larger microbes such ...or bacteria, mycoplasma can be extremely hard to detect with levels reaching 108 cells per ml before the...or PCR-based approaches to detect even low levels of contamination. For those labs that prefer a more ...more economical mode of detection, Uphoff et. al. outline a do it yourself PCR-based approach complete with...alters microarray gene expression profiles.” Biotechniques 35.4 (2003). 812-4. Pubmed PMID: 14579747. 3...

...Sample preparation for these techniques sometimes makes it more difficult to detect protein tags using antibodies...availability of reliable primary antibodies for their detection, particularly when antibodies to the POI are non-specific...multimeric tags are frequently insufficient for detection when the POI is weakly expressed. As these epitopes...have no intrinsic visual signal, they cannot be detected directly in live imaging experiments. It is therefore... are both intrinsically fluorescent and can be detected with highly epitope-specific, fluorescently-labeled...can be easily expressed in the brain and can be detected through their direct fluorescent signals or using...antibody binding sites and primary antibody-based detection, smFPs create much brighter signals for weakly...