...protein-only transduction method and its potential applications to CRISPR/Cas9 genome editing.   Protein-only...the HIV protein Tat. Although CPP fusions can translocate a cell membrane, the presence of the CPP may ...that components in the buffer used for protein purification must be helping the untagged protein enter cells...Diego D'Astolfo. CRISPR/Cas9: a potential application of iTOP Having tested iTOP using Cre-lox recombination...showed up to 70% resistance to diphtheria toxin, indicating a 70% frequency of biallelic mutations. Importantly...Importantly, hESCs retained their stemness, indicating that iTOP may be valuable for CRISPR/Cas9 stem cell...potential for off-target effects. Third, protein purification (once a protocol has been established) may be...

...Gibco organizes its app into four categories: tools, analytics, education and data manager. The tools are...are five FREE science lab apps that have been educational and great for lab life. These apps prevent you...lab notebook. Lab Archives is a multipurpose application that allows you to share your data with colleagues...other nice features. This app also has a great educational section. This section holds the Gibco Cell culture...directions including which buffers to use and that indicates if a sequential digest is needed. The enzyme finder... to this app; the user can search by sequence, category of enzyme, or name to discover all the information... Addgene.org Find Protoctols for Your Research Catch Up On Your Molecular Biology Basics ...

...together a grant application? Are you familiar with the publishing or patent application processes? How ... pages in a Curricula Vitae to showcase your publications, academic performances, and research experiences...information. Think "skills," not just "research and publications” What are the skills you developed through your...and writing was how I got my current job as a Communications Coordinator. Wondering how you can develop ...So You Think You have Skills.   Get Science Communication Practice with a Student Group Like Science in...Blogger! Theresa Liao (@theresaliao) is the Communications Coordinator for the Department of Physics &... a research career, she transitioned into a communications role instead after she completed her MSc in...

...your application a whole week before the deadline. The day of submission, review your application with... their dream.  To help students focus on the application process, and simplify access to academic resources...Scrutinize every opportunity prior to starting an application to assess your eligibility, interest, and career... detailed. Beyond the resume, a cover letter communicates your interest in a specific program, and institution...February represent peak season for internship applications. Thus these noble individuals are inundated ... with a stellar letter of support.        5. Application submission  Set a calendar reminder for every...about outreach among low-income communities to educate parents about resources that can help their children...

...ITRs serve as a type of origin of replication to initiate replication of the dsDNA virus. Viral packaging...plasmid while the other plasmids with packaging or replication components are called helper plasmids. Below,... production, they can be helpful for certain applications and aid in ease of use. Our lentiviral guide...AAV are rep and cap. Rep is required for viral replication and also aids in infrequent genome integration...a helper plasmid and mediate efficient viral replication. More information on these viral genes is available...particles. E2A/E2B are responsible for viral replication, with E2B being the primary viral polymerase....is missing the endogenous E1 gene, making it replication deficient. This is typically supplied by the ...

... its function, mimic a biologically relevant modification, or add in fluorescent or chemically reactive...function relationships or, in more practical applications, allow a protein to be more effectively used... you do to any number of experiments with. The catch The rapid expansion of GCE led to the creation of... of many new tools and potential applications. But this focus on development, along with the inherent ...’t give it a try. Optimization Why is GCE so complicated? Well, while the techniques are straightforward...ligation through introducing ncAAs with protein modifications of interest or any of a huge variety of ncAAs...has “10x the impact of PCR. But it’s 100x more complicated.” And Ryan Mehl would know. He’s the director...

...Our educational content  Have you ever wondered how we choose topics for the Addgene blog? Or why we...everything else we do! The goal of Addgene’s educational content Addgene truly believes that sharing speeds...antibody and content team, we identified what applications should be included and what basic information... The guide was then assigned to our science communication specialist, Rachel, to write. Identify expertise...through an editing process and final check before publication.   Do research! Nearly everything we write entails...Guide figures. This requires a fair amount of communication between our content team and our graphic designer...graphic designer upon request that the science communicator "just sketch a western blot out quickly." Final...

...discuss automated protocols. 12:00 PM--Catch up. Finish any communications with my UK team members before they...getting to talk to so many different people! I communicate with customers, software developers, as well ...from interacting with others, and have strong communication skills to tailor your message to any type of... to solve is clear, but the solution is more complicated. You’ll need to be creative to be able to work...team in the UK is five hours ahead, so I’m always catching up in the morning! 9:00 AM--Technical implementation...out. 4:00 PM--Respond to customer help tickets. Catch up on customer help tickets, and respond when I ...technology, software, and science. She loves to advocate for the customer and help build software and technology...

... every genomic locus, and to do so in vivo. Catalytically dead Cas9 (dCas9) cannot induce gene editing...Cas9 ortholog, and the target sequence must be located adjacent to that PAM. To scale up fluorescent CRISPR...this rainbow of colors, Ma et al. performed sophisticated tracking of multiple chromosomal loci in live... do not induce genome editing. If used with catalytically active Cas9, the short, CRISPRainbow gRNA will... PMCID: PMC3918502. Find plasmids from this publication at Addgene. 2. Ma, Hanhui, et al. “Multicolor...PMCID: PMC4364232. Find the plasmids from this publication at Addgene. 3. Ma, Hanhui, et al. “Multiplexed...PMCID: PMC4864854. Find the plasmids from this publication at Addgene. 4. Kiani, Samira, et al. “Cas9 gRNA...

... antibodies may be indicated on occasion, usually after other signal amplification techniques have been...IgG2 or IgG4. This can be important for some applications. Assay use While secondary antibodies are useful..., the flexibility of their use varies across applications. Western blots, for instance, almost always ...For the latter three, secondary antibodies are indicated when the protein of interest is found in low amounts...been ruled out.  Ease of access Signal amplification isn’t the only benefit of secondary antibodies. Finding...quantitative analysis of a sample. Instead, relative quantification can be done using either the direct or the ...antibodies remain the go-to option for signal amplification while providing needed flexibility in assay ...

...from the serum. Protein A/G purification Protein A or Protein G purification is one common method. These...reagent that works for your species. Affinity purification Only up to 5% of the IgG antibodies produced...specific for the immunogen. A different type of purification can be performed to isolate antibodies only ...inexpensive. Their short production time and the bulk purification steps keep the cost low compared to other antibody...are particularly useful for certain research applications. Many secondary antibodies are polyclonal, as...target protein thereby greatly increasing signal amplification.  When to stay away from polyclonal antibodies...2018-0065 Fishman JB, Berg EA (2019) Antibody Purification and Storage. Cold Spring Harb Protoc 2019:pdb.top099101...

...thing that comes to mind. However, ion-based communication is not just for neurons. Non-neuronal cells ...It's just that for non-neural cells, ion-based communication happens at a slightly slower scale.  All living..., and when gap junction-mediated electrical communication is briefly inhibited in the entire worm during...reporting and genetically encoded fluorescent voltage indicators (GEVI) are ideal tools to track bioelectric states...activated proton pump) and ChR2 (blue-light activated cation channel) light-activated channels to hyperpolarize...channelrhodopsin used as a sensitive fluorescent voltage indicator with near-complete desensitization in continuous...extremely exciting emerging field, with many implications for regenerative medicine, evolution of body...

... work around this is to "domesticate" your fragment: PCR-based amplification can be used to create silent...and exciting applications, this system is an incredibly powerful tool for cloning complicated constructs...Type IIS sites (such as BsaI or BbsI), that are located on the outside of the cleavage site. As a result...plasmid (Option 1) or added using PCR-based amplification (Option 2). Advantages of Golden Gate cloning...restriction sites that may not be amenable to "domestication", you might want to consider using an alternative...high-efficiency step. References: Sequential amplification of cloned DNA as tandem multimers using class-IIS...

...mammalian cells often relied on chemical (CaCl2, cationic liposome) or electrical (electric current forming...particles and the virus particles produced could not replicate. However, Mother Nature found a way to re-create...packaging plasmid forming a vector that could create replication competent virus. Even though this system was ...still a potential risk for the generation of replication competent lentiviruses (RCL) especially if you...factors in HIV positive cells could make the virus replication competent. The 3rd generation of lentiviral...transgenes (>8 kb) Potential for generation of replication-competent lentivirus (RCL) Efficient gene transfer...transfer Potential for insertional mutagenesis: Replication-incompetent lentiviruses with human tropism can...

...promoter, such as the truncated tyrosine hydroxylase (TH) promoter for catecholamine neurons or glial fibrillary... Flp), to either flip or excise a gene that is located between them. Early recombinase-dependent vector...expression profile at multiple steps from plasmid amplification in bacteria to transgene expression in vivo.... the level of spontaneous recombination. This indicates that the amount of leaky expression can largely...are typically employed during viral plasmid amplification (Fischer, KB. 2019). This means that all DIO...of spontaneous reversion during AAV plasmid amplification. On the left homologous pairs of scrambled cre-recombinase...expression of receptors, post-translational modifications, and other factors that AAV utilizes to gain...

... a calcium sensor Genetically encoded calcium indicators (GECIs) are fluorescent proteins linked to calcium...in living cells and organisms. Unlike chemical indicators, these DNA constructs can be delivered to intact...the sensors discussed below fall into this GECI category.   For calcium sensors, one size doesn...:509-18. Inoue M. Genetically encoded calcium indicators to probe complex brain circuit dynamics in vivo...Kanemaru K, Iino M. Genetically encoded fluorescent indicators for organellar calcium imaging. Biophysical journal...neuronal activity with genetically encoded calcium indicators. Cold Spring Harbor Protocols. 2012 Jun 1;2012...signaling using genetically targetable fluorescent indicators. Nature protocols. 2006 Aug;1(3):1057-65. Resources...

...host genome, the virus is replication incompetent, meaning it can’t replicate and continue the infection... tools, thought was given on how to keep the replication of virus and its infectivity in check. Engineering...envelope plasmid), and the polymerase (Pol) for replication and Gag structural protein on yet a third plasmid... this safety feature prevents the virus from replicating and spreading in non-target cells, including ....    Viral cargo-specific safety   After replication competency, the second concern with virus safety...diseases in humans, earning this virus a BSL-2 classification. Lentivirus (a type of retrovirus) and gamma...decontaminating liquid waste, remember that the percentage indicates the proportion of the decontaminate in the waste...

...another method for making targeted modifications. A recent publication by Zhang et al. shows just that -...about a month and a half, without the need for complicated cloning of homology arms. MMEJ: An Introduction...required by MMEJ can easily be added via PCR amplification. Given the inefficiency of HDR for knock-ins... and 3’ junctions, respectively. These results indicate that PITCh is a robust method for genomic insertion...lies in the diversity of nucleases and their applications. It will be interesting to see what new editing...PMID: 26678082. Find the plasmids from this publication at Addgene. 2. Dalvai, Mathieu, et al. (2015...633. PMID: 26456817. Find plasmids from this publication at Addgene. 3. Zhang, Chi, Xiuhua Meng, Xiaolei...

...well as suitable applications. Expression System Components of System Application Mammalian expression...CRISPR mammalian expression systems, the typical applications for each, and potential delivery methods.   ...Select the appropriate CRISPR tools for your application: Wildtype Cas9 or Cas9 nickase are appropriate... or introducing mutations and tags, while a catalytically dead dCas9 can be used in conjunction with activator...or more sgRNAs and Cas9 are required for any application. If you want to include a homology directed repair...Cas9-gRNA complex and delivered to cells using cationic lipids. Transient expression of CRISPR components...below: Chemical transfection   Lipid mediated Cationic polymers Calcium phosphate Cells Transformed...

...up. Applications of photoactivatable fluorescent proteins Some of the most important applications for ...conventional FP imaging. PA-FPs are divided into 2 main categories - those with irreversible and reversible photoactivation...photoactivation - and have enabled sophisticated imaging techniques.     Irreversible photoactivation Irreversible...level of background fluorescence. For any PA-FP application, the brighter the fluorescence the better. More...photoactivation. A high contrast ratio usually indicates that a PA-FP has a low level of spontaneous fluorescence...photostable green and red states. Beyond these imaging applications PA-FPs have been developed for use in yeast,..."Photoactivatable genetically encoded calcium indicators for targeted neuronal imaging." Nature methods...