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...beta-barrel structure. Although the beta barrel has no strong affinity for other proteins, it may still block...There are several ways to measure stickiness. In vitro, ultracentrifugation or gel filtration is the most...Avoiding protein over-expression artifacts The introduction of DNA that encodes a fusion protein adds protein...knock-out of the corresponding protein, that is strong evidence that the fusion behaves well. But this...fusion proteins: random insertion of GFP with an in vitro transposition reaction." BMC neuroscience 3.1 (2002...

...which refers to mEGFP. This variant was made by introducing two mutations (F64L, S65T) in the original jellyfish...fluorescent proteins can be accurately determined in vitro by measuring the fluorescence intensity of purified...Peptide-tags for site-specific protein labelling in vitro and in vivo." Molecular BioSystems 12.6 (2016): ... "Blue fluorescent amino acid for biological spectroscopy and microscopy." Proceedings of the National...Additional Resources on the Addgene Blog Read our Introduction to GFP Read our Fluorescent Proteins Blog Posts...

...least 3 months experience working in a developing tropical country. This is why I set up a placement working...very hard to speak with individuals who had very strong beliefs in traditional medicine, especially when...very new, and everything was more structured and controlled. I worked from Standard Operating Procedures ... reliant on programming skills which are not my strong point. Working in industry: virology On completing...because it is an English speaking country with a strong economy and plenty of jobs in science at world ...

...lentivirus envelope plasmid pMD2.G from Didier Trono’s lab! Lentivirus envelope vector pMD2.G This plasmid...expresses the VSV-G envelope protein under the control of the powerful mammalian CMV promoter. Envelope...protocols and information, be sure to check out the Trono Lab website. If you have plasmids you’d like to ... Guide to All Things Lentivirus 5 Tips for Troubleshooting Viral Transductions Getting the Most from Your...

...offer serotypes AAV1, AAV2, AVV5, AAV8, or AAV Retrograde (AAVrg), and volumes of 1 mL, 2.5 mL, or 5 mL...necessary MTA, then start production and quality control.  Our packaged on request AAV service uses the ...the same trusted production and quality control processes as our in-stock service: ddPCR, purity testing...blog post on viral vector production and quality control, our AAV Packaged on Request webpage, or our announcement...

... AAV9 Controls Jensen New serotype pAAV-CAG-2xNLS-tdTomato-WPRE-hGHpolyA AAV9, PHP.eB Controls Feng ...from the Gradinaru lab. This is useful for both in vitro and in vivo work with many cell types, as CAG is...serotypes pAAV-hSyn-GRAB_OTmut AAV9 Biosensors (Control) Li New viral service pAAV-hSyn-GRAB_ACh3.0 AAV9...

...FITS In vitro CRISPR screens in immune cells have identified many putative regulators of immunity. However...However, in vitro screens cannot faithfully model the complex spatial, cellular, metabolic, and cytokine...modified cells; and (5) is compatible with the introduction of unique molecular identifiers, which can improve...includes pLX_305_Ovalbumin, a plasmid that enables introduction of the model antigen, Ovalbumin, into cell lines...

...m an introvert!” is not an excuse. Every psych test I’ve ever taken has declared me an introvert, and ...Scientist who coordinates all kit creation and quality control efforts with depositors; Gizela, the lab member...deposits into the repository, performing quality control, and providing technical customer service. Team...easier. Trust Ultimately, your team could have a strong sense of purpose, an excellent leader, and perfectly...

...Transfection grade DNA Figure 1: Transformation introduces transformation grade plasmids into a bacterial...bacterial transformation of chemically and electrocompetent cells is minimal (picogram range). It can ...2.2, and provide an indication of purity versus chaotropic agents as guanidine thiocyanate and guanidine...Transfection grade DNA Figure 3: Need to introduce plasmids into mammalian cells? Use transfection...

... other projects, using stem cells to develop in vitro models for lung disease. Darrell strives to promote...the interview with Darrell Kotton Chapter 1: Introduction to Darrell Kotton and the Boston University ...reliable projects. Chapter 9: Using stem cells as in-vitro models of disease. How Darrell is using stem cells...helping us stick to our core mission. Chapter 13: Outro - remember to step back and ask, "Why did I start...

...positive selection (ex: antibiotic selection and auxotrophy) and negative selection (ex: toxin-antitoxin ...technique from David Liu’s lab that gives more control and flexibility to CRISPR editing. Using a fusion...transcriptase and a prime editing guide RNA, it can introduce insertions, deletions, and all possible base-to-base... and Transfection by Alyssa Cecchetelli The introduction of nucleic acids into bacteria or eukaryotic...

...any nascent technology, researchers are having trouble getting NgAgo genome editing up and running in ...get a better idea of how many people are having trouble, to determine if there have been any early successes... users likely need more time to optimize and troubleshoot the system. Site Directed Mutagenesis by PCR... with Enhancers and Epigenomics 5 Tips for Troubleshooting Viral Transductions Single Base Editing with...

...can easily spot the colors and text. Tip 2: Use electronic to-do lists to access your tasks on the go Never...could easily lend one to another labmate? Though electronic lab notebooks (ELNs) have gained traction with...to borrow most frequently. Copy these into an electronic file sharing tool, such as Google Drive or Dropbox...documents from anywhere, but also allow you to control who can edit the documents and track the revision...

...Cas9 (dCas9) or a Cas9 nickase (nCas9) to avoid introducing double-strand breaks. The only things base editors..., the team noticed that this editor sometimes introduced C → G edits at certain positions within the editing...during cytosine-to-uracil editing, and instead introduced its opposite, a uracil DNA N-glycosylase (UNG...end joining (NHEJ). To mitigate this, the team introduced the enzyme HMCES, either as a fusion construct...ever. References and Resources Glossary We introduced a lot of terminology in this post! Here’s a quick...

... organs located deep in the body, like the gastrointestinal tract. Require Administration of Substrate...pharmacokinetics and biodistribution therefore introduce additional confounding factors when attempting...reporter genes for in vivo imaging Following in vitro characterization, Bourdeau et al performed a proof-of-concept...coli aided in ultrasound imaging of the mouse gastrointestinal tract. The E. coli strain Nissle 197 (ECN)...

...it is non-invasive and offers a high degree of control over time and space. Zebrafish have become a popular...are not native to vertebrates and are commonly introduced in zebrafish through the UAS/Gal4 system for ...had previously been a mystery, but through the control offered by the optogenetics toolbox and the versatility..., Filippo Del Bene, and Herwig Baier. "Optical control of zebrafish behavior with halorhodopsin." Proceedings...

...serotype-specific differences in tissue and/or cell type tropism. Introducing changes and/or short peptides sequences ...variable loops is one method for changing a capsid’s tropism.  cap also encodes the Assembly-Activating Protein...References: Naso MF, Tomkowicz B, Perry WL III, Strohl WR (2017) Adeno-Associated Virus (AAV) as a Vector...

...range from researching an area, to writing an introduction that gives a basic overview of kinematics in... to students, like how to pour a gel [for] electrophoresis or doing a dissection video. I did that in ...traveling with the sales team to meet faculty and introducing the kits to clients who might initially be doubtful...themselves. “You open up the box and there’s a gel electrophoresis tray in there. Or the dissection specimens....

... eSpCas9 and SpCas9-HF1. By mining the treasure trove of naturally occurring Cas9 diversity via the Integral... Table 1. Comparing SpCas9 and GeoCas9. *For in vitro Cas9 RNP activity or longevity. GeoCas9 is active...Besides being able to take some heat, GeoCas9 can introduce genomic edits in mammalian cells. Harrington et...GeoCas9’s thermostability could also be used with in vitro molecular bio applications that require high temperature...

...          Special Thanks Trophies and engraving services from Hit Trophy Additional Resources on the...positively affect scientific research. As Maria Castro of University of Michigan says, “Addgene has transformed...distributed more than 100 times. We hope these desktop trophies will serve as badges of honor for our powerhouse...

...translated into proteins, or, in the case of retroviruses, reverse transcribe their gRNA into DNA, which...is. Thankfully, through the use of engineering controls — changes made to the virus that eliminate or ...viral vectors to reproduce only in experimentally controlled conditions, making them far safer to work with...they’re derived from: AAVs, lentivirus, gamma retrovirus, and adenovirus. And now that you know what viral...

...epitope tags? Epitope tags are short peptides introduced at the N or C terminus of a protein that are ...hundred amino acids, depending on the tag, and are introduced via knock-in at a genomic locus or more commonly...should be used with caution or not at all. The introduction of a tag at the N or C terminus of a protein...  Susanna Stroik...

...group optimized their initial system by using a stronger promoter and different biochemical secondary energy...cell free system allows for a relatively simple, controlled “one-pot” reaction capable of reproducing useful...codons, splice acceptor and donor sites, and introducing premature stop codons.    New from the viral... and 5HT sensors New AAV tools to selectively control parvalbumin interneurons from the Dimidschstein...

... promoters are upstream or 5’ of the gene they control. Promoters can be organism or tissue type-specific...different levels of expression. For example, CAG is a strong promoter that drives very high, ubiquitous expression...recombinase, are used to spatially and temporally control transgene expression. Lox sites are directional...availability of Cre, lox sites lets researchers control the spatial and temporal activation of a transgene...

...PubMed PMID:29109157. Nanobody toolkit to monitor retrograde transport at the cell surface Article contributed...developed a set of functionalized nanobodies to study retrograde transport from the cell surface. Their toolkit...treated with biotin–phenol and H2O2, APEX2 labels electron-rich amino acid side chains (e.g. Tyrosine) with...biotin-labeled proteins can them be analyzed by mass spectrometry allowing a researcher to specifically identify...

...increased yields in adenoviral (Coroadinha, 2006) and retroviral production systems (Shen, 2011). To our knowledge...also improved yields of the AAV2 derivative, rAAV2-retro, but had no effect on AAV5 yields. We further show...preparation. Through sugar detection assays, in vitro transduction, and silver stain approaches we show...Effect of osmotic pressure on the production of retroviral vectors: Enhancement in vector stability." Biotechnology...

...chemically or electro-competent E. coli - excess salt causes arcing during electroporation which would ...sequenced for potential positive clones. Once you introduce your 3’-end “A” overhang insert, the magic of ...would cause the electroporation to fail). When incubating at room temperature, it is not recommended that...

...gene-editing protein - a DNA-cleaving Argonaute from Natronobacterium gregoryi, or NgAgo for short. Addgene has ..., showing that this protein could cleave DNA in vitro, directed by phosphorylated DNA guides. Unfortunately...RNA guides must be expressed from a plasmid or in vitro transcribed, and changes in gRNA secondary structure... Han. “DNA-guided genome editing using the Natronobacterium gregoryi Argonaute.” Nat Biotechnol. Epub ...

... 10. Plasmids 101: Control Plasmids Need help thinking about the types of controls you’ll need for your...provides researchers with many different ways to control gene expression. Get the full scoop from Addgenie...Addgenie Chari Cortez guides us through some common controls and why you might need them. Happy Reading!   ...

...pairs from intergenic regions, intronic regions, and a pericentromeric region. All pairs were easily visualized...CRISPRainbow. Both tools were targeted to the FBN3 intronic repeat which contains 22 copies of the gRNA target...locations of the subtelomeric region of the pericentromeric region (PR1), the p-arm (T1), and the q-arm...

...abstracts comparing the mortality rates between two controversial health issues. One group read the text from ...design or by their preconceived ideas about the controversial medical issues. Participants gave significantly...methodologically flawed, and biased. As a result, the control abstract comparing two fictional medical issues...and education have more polarized beliefs on controversial science topics. Proceedings of the National ...

...Xin hypothesized that she could make enzymes controllable by tethering them to Dronpa, a fluorescent protein...their ability to be precisely spatio-temporally controlled as a key advantage over other inducible enzyme... sugars (including lactose), and a lac operon controlled GFP. When the enzyme breaks down the HMO of interest...Photosactivatable Fluorescent Proteins Use Light to Control CRISPR Genome Editing Choose the Brightest Fluorescent...

...The gRNAs targeting EGFP can be used as positive controls for gene editing or gene activation, while the...What you don't need to do is spend your time troubleshooting reagent production, because that's a distraction...from your primary goal. Getting these quality-controlled reagents from Addgene can greatly expedite a ...Research: A Primer for Beginners 5 Tips for Troubleshooting Viral Transductions Additional Resources on...

...later, it was shown that putting the operon under a strong promoter results in high R-body expression  (Schrallhammer...bodies could also perform as actuators in microelectromechanical systems (MEMS) devices. They could be functionalized...of armor to protect their host from predation (Petroni et al 2000). With a better understanding of how... Plasmids from This Publication at Addgene 4. Petroni, G., Spring, S., Schleifer, K.-H., Verni, F. & ...

...unique editing tasks, classic Cas9 nuclease, which introduces double stranded DNA breaks, can be expressed ...Gαi-coupled) DREADD fused to mCherry, all under the control of the human synapsin promoter. Purpose of this...mCherry can be used to induce neuronal silencing in vitro and in vivo (Krashes et al., 2011). The various ...Rumpel S. Analysis of transduction efficiency, tropism and axonal transport of AAV serotypes 1, 2, 5, ...

...digest as depicted in Lane 2. Controlling methylation Finally, you can control methylation by altering your...only the non-mutated and methylated template is destroyed leaving behind a pool of mutated plasmids which... with the Latest Cloning Techniques Learn to Troubleshoot Your Sequencing Results Use REPLACR Mutagenesis...

...antibodies. The introduction of antibody fragments, such as nanobodies, has introduced new options for...Antibodies 101: Introduction to Immunofluorescence Antibodies 101: ELISAs Antibodies 101: Introduction to Antibodies...

...antibody, you’ll want to use the same positive control across experiments to be aware of signal strength...you’ll want to be sure to choose the appropriate controls to be sure the staining you are seeing is real...real. It’s important to include a negative control using serum from the same species your polyclonal antibody...antibody was generated in or a polyclonal isotype control, to account for nonspecific binding. Now that you...

...localization. Surprisingly our observations at the electron microscope showed us that, in monkey neurons, ...of the haemagglutinin of the influenza virus, introduced to the field of fusion proteins more than thirty... of interest the membrane.   Figure 1: Electron microscope images showing ultrastructural localization...protein of interest, an alternative could be bicistronic expression of the fluorescent protein with the...

...the insert. Gateway® technology (developed by InvitrogenTM) is essentially a more modern version of this...teachline.ls.huji.ac.il/72682/tutorials/gateway/introduction.html demonstrating the principal behind the “swapping...CcdB-resistant strain is ccdB Survival™ from InvitrogenTM. Using either DB3.1 or ccdB SurvivalTM, one ...PubMed PMID: 1324324. 6. Tam, JE. and Kline, BC. “Control of the ccd operon in plasmid F.” J Bacteriol. 1989...

...AAV (AAV-pgk-Cre) has been used to successfully introduce Cre into cells of interest. What if I need two...of using loxP sites is the inability to tightly control which loxP sites recombine if more than two are...allowing for two separate recombination events to be controlled in parallel. Have you used the Cre-lox system...PubMed Central PMCID: PMC49604. 4. Independent control of immunoglobulin switch recombination at individual...

...chemogenetics? Chemogenetics relies on genetically introducing engineered receptors that are only activated ...Expression Using chemogenetics requires successfully introducing receptors to target cells. Targeted and specific...mitigated by using low doses and running appropriate controls (Jendryka et al., 2019), they have resulted in...channel, the PSAM acts as a gating mechanism to control ion flux. The system allows you to directly manipulate...

...live cells.  Controls  When designing experiments, it’s important to include proper controls to assess the...quantitative readout of the interaction in vivo or in vitro, and can be used for measuring protein expression... p53 tetramerization in response to DNA damage controls the p53 response. Gaglia et al. Mol Syst Biol....

...of interest to better study them in vivo and in vitro, especially in the mid-late 20th century with the...reticulum localization, Munro and Pelham casually introduced the Myc-tag (Munro, et al. 1986; Munro and Pelham... and the Myc-tag today Since Munro and Pelham introduced the Myc tag to the world, it has become one of...this antibody works and allow users to better troubleshoot and design experiments. It turns out that even...

... proteins. The Seeger lab developed a robust in-vitro protocol that enables rapid selection of synthetic...sybodies that hypothetically contain ones that bind strongly to your target protein. The Seeger lab designed...selection step involves using a commercial kit for in-vitro translation of the mRNA libraries. Each mRNA molecule...enrichment step for identifying sybodies that bind strongly and specifically to the protein of interest. The...

...advantages degron tags can offer over other protein control methods and compare the commonly available types...etc. Firstly, they are reversible mechanisms of control. Protein degradation can be induced by the addition... Tsien RY, Lin MZ. Tunable and reversible drug control of protein production via a self-excising degron...  Susanna Stroik...

...can be exploited to either inactivate a gene or introduce a desired genetic alteration. Listen to Our Podcast...necessary gRNAs, but it would not be possible to control which gRNA becomes complexed with which variant...represents a straightforward means of exerting tight control over Cas9 nuclease activity while eliminating the...guides targeting one set of genetic loci can be introduced together with truncated gRNAs targeting a different...

...experiments attempting to build gene drives in tropical mosquito vectors of diseases such as malaria and...then, using multiple confinement strategies is strongly advised. The effects are multiplicative, and there.... Site-specific selfish genes as tools for the control and genetic engineering of natural populations....Fitz-Earle, M., Holm, D. G. & Suzuki, D. T. Genetic Control of Insect Populations: I. Cage Studies of Chromosome...

...and would avoid genomic off-targets or indels introduced through non-homologous end joining (NHEJ). Designing...recapitulate or rescue known pathogenic alleles, or introduce a premature stop codon to render an RNA nonfunctional...also adjusted the structure of the guide RNA by introducing a C opposite the target A to be edited. This ...off-target edits around the targeted nucleotide. Introduction of guanine-mismatches in the guide across from...

...for Laboratory Automation and Screening (SLAS) Electronic Laboratory Neighborhood.   Work/life balance....possible to bring all aspects of our lives under control? Balance is a myth and should not even be the real... don’t have children, so my life will be under control.” Think again. Maybe you’ll need to care for an...to do most tasks – try to resist the impulse to control by insisting things always be done your way. Be...