...this versatile technique.  Immunoprecipitation Overview An immunoprecipitation reaction is typically carried...Immunoprecipitation (IP) uses immobilized antibodies, or immunoglobulins, to isolate a specific protein...primary antibody Immobilization is the key to immunoprecipitation and simply refers to the process of anchoring...

...producing AAV vector preparations, we have used quantitative PCR to evaluate the physical titer of our preps...digital PCR (ddPCR) comes in. AAV titering using quantitative PCR Before we dive into the details of ddPCR...ddPCR, we should first note that quantitative PCR (qPCR) has been a powerful tool for quantifying AAV for...

... you. Statistics – The basics Statistical analysis is what determines if there is statistical significance... several core factors of statistics relevant for many downstream statistical tests.   Independent variables...are statistically different. They are classic parametric tests and one of the simplest statistical tests...described above. Violated assumptions and statistics Statistical power, the ability to correctly identify... non-parametric statistical tests! Adding it all up  There are many, many statistical tests beyond the...your friendly neighborhood biostatistician happy by making your statistical analysis plan when you design...Wissing, Dennis R., and Timm, Donna. Statistics for the nonstatistician: Part I. South Med J., 105(3): 126...

...Addgene plasmids get more citations So what do we find? Lots more citations for the papers with plasmids... difference in citations after controlling for journal. And so, while our quantitative analysis cannot...editing paper on CRISPR/Cas amassed nearly 2,400 citations in its first four years (1). In addition to publishing...associated with Addgene deposits accumulate more citations than those without Addgene deposits? Even more...network of articles to track the total number of citations the paper receives. From the database we pulled...papers published in other years also get many more citations This isn’t the whole story, though. It would ... great to know how much of that difference in citations is caused by depositing a plasmid with Addgene...

..., increasing overall packaging capacity but necessitating purification and co-infection of two AAVs. ...

... Marc Zimmer’s recent book, The State of Science: What the Future Holds and the Scientists Making It ... ago. Teaching by example No examination of the state of science would be complete without the inclusion... last decade. He describes the detection of gravitational waves--a story I was much less familiar with...to. The problems and challenges described in The State of Science have not changed in “COVID times,” but...next week.   A stepping stone When I discussed The State of Science with the five Addgene colleagues who ... learn everything about one specific topic, The State of Science could be a jumping off point to explore...

...the plasmid here yet? A quick look at your order status on addgene.org identifies the hold-up. Your Material...possible new awardees, and we’re happy to share stats and strategies with tech transfer offices. Drop ...Sloan-Kettering Cancer Center  Monash University  Montana State University  National Institute of Diabetes & Digestive...

...ready for deep mutational scanning.     3 reasons to be interested in deep mutational scanning Hundreds...effect of multiple mutations in combination and possibly discover unexpected epistatic changes in protein...combination of library coverage, mutational efficiency, control over where mutations are introduced, and scalability...still work if the orientation of the BbvCI site(s) of the gene are in the same orientation as those in the...Scientists use deep mutational scanning to simultaneously test how multiple amino acid changes affect...link genotype to phenotype that can be used with mutational scanning: ligand binding assays, phage display...mutagenesis Several methods can be used to create mutational scanning libraries. While each of these methods...

... Commercial DNA Ladder Penn State Ladder: Homemade Plasmid Prep Penn State Ladder: Commercial Plasmid ...undergraduate students led by Dr. Song Tan at Penn State developed the plasmids, pPSU1 and pPSU2. When restriction...$200-$500 for 1000 lanes worth of ladder. The Penn State ladders are significantly cheaper. When using a ...

...Collection for “Functional Annotation of Somatic Mutations in Cancer.” Of all the mutations and fusions represented...attempting to characterize mutations in a pooled cell population, as mutations with strong phenotypic effects...types of mutations they identified included gene amplifications and fusions, missense mutations, and indels...assay allows first-time functional annotation of thousands of mutations The researchers used the collection...Kwok-Shing, et al. "Systematic functional annotation of somatic mutations in cancer." Cancer Cell 33.3 (2018)...Thousands of mutations have been implicated in cancer. Yet, the majority of them remain uncharacterized...moderate throughput functional genomic platform to annotate genetic perturbations that may play significant...

...your research interests. We do this by promoting citation, supporting sophisticated search, and curating...reference for its original publication. To help facilitate this process, we explain how to cite plasmids...Addgene right on our plasmid pages and provide a citation that you can put straight into your reference ...visibility of both you and the original depositor. Our citations feature makes it so that, when you cite a plasmid...quick note -  While it is tempting to rely on the citation number when comparing two plasmids, we recommend...Impact Are Evolving Check out the Blog Post on Our Citations Feature Browse More Inside Addgene Posts Additional... citation...

...Recombinase-based state machines (RSMs), which take on a particular DNA-sequence configuration (state) based on...effects on the state cannot be undone and also limit the set of possible downstream states that can be achieved...order of inputs results in only one final state, then the state of the RSM will tell you precisely what ...might one read the state of an RSM? The most straightforward way to read the state of an RSM is to sequence...is to interrogate state with qPCR. The re-arrangement of DNA segments in each state of a register enables...certain states of interest. For example, in Figure 2 below we present a two-input, five-state GRSM register...green fluorescent proteins in each state. The three methods of state interrogation mentioned above (Sanger...

...Bimolecular Fluorescence Complementation (BiFC). What is bimolecular fluorescence complementation? Bimolecular fluorescence... TK (2006) Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the... fluorescence complementation is a method for detecting protein-protein interactions that uses split fluorescent...  Applications for bimolecular fluorescence complementation BiFC has been used to directly visualize PPIs...success of BiFC experiments. The ideal control is a mutated variant of one or both of your PIPs in which the...Living Cells Using Bimolecular Fluorescence Complementation. Molecular Cell 9:789–798. https://doi.org/...Kerppola TK (2013) Bimolecular Fluorescence Complementation (BiFC) Analysis of Protein Interactions in ...

...wildtype 5'LTR, which requires the presence of the Tat protein to work, while 3rd-generation transfer plasmids...chimeric 5'LTR removes the requirement for the HIV Tat protein, thus decreasing the probability of creating...plasmid encoding HIV proteins gag, pol, rev, and tat. 3rd generation systems require an envelope plasmid...transfer plasmid to eliminate the need for the HIV Tat protein and consequently are considered safer. Second-generation...system because the wildtype 5'LTR promoter requires Tat to function. Third-generation plasmids can be packaged...Second-generation contains those three genes plus tat. The envelope plasmid provides a heterologous envelope...biosafety available for the technology. The minor limitation in using the 3rd-generation system is that it...

...virus. Second, Tat is eliminated through the addition of a chimeric 5’ LTR containing a Tat-independent ...polymerase), Tat (viral transactivator for transcriptional activation from the 5’ LTR) and Rev (facilitates nuclear...transfer plasmids. The packaging plasmid carried a mutated HIV-1 provirus that couldn’t package itself due...envelope plasmid contained a viral envelope to dictate the tropism, or types of cells the vector could...flanked by HIV-1 long terminal repeats (LTRs) that facilitate viral packaging and host genome integration. ...

...for the production of a viral particle (Gag, Pol, Tat, Rev and Env) and the transfer plasmid encoding the... a clinical setting. In this generation, the HIV Tat gene (previously used to drive expression from the... has been inserted in this LTR so you don’t need Tat to transcribe the viral genome in producing cells...the LTRs) has been removed, Rev (which facilitates nuclear export) is expressed from a separate plasmid...active area of research due to the possibility that mutation or recombination with human retroviruses could... In this case, your gene is repressed in steady state and is expressed when Dox is added to the culture...centrifuge the culture supernatant in order to precipitate the viral particles. Some protocols suggest adding...

.... One such CPP was discovered in the HIV protein Tat. Although CPP fusions can translocate a cell membrane...toxin, indicating a 70% frequency of biallelic mutations. Importantly, hESCs retained their stemness, indicating... remove the risk of viral integration causing mutations and gene expression changes elsewhere in the genome..., working directly with proteins removes the limitations of packaging size associated with viral vectors...

...longer requires the viral transactivator, Tat. For more information on the different generations of lentiviral...promoter of the 5’ long terminal repeat (LTR) is mutated in third generation systems such that it no longer...spin columns, and polyethylene glycol (PEG) precipitation. All of these methods ultimately isolate the...downstream applications. For example, while the PEG precipitation method is relatively inexpensive and does not...not require an ultracentrifuge, PEG will precipitate all proteins in a solution, not just the lentivirus...

...Figure 1: A) Cell-penetrating Cas9, fused to HIV TAT, Myc and SV40 Nuclear Localization Signals, and GFP...co-incubate it with an “assist peptide” that facilitates endosomal escape. Combining Cas9-PAGE with lentiviral...Balasubramanian and colleagues showed the single mutation E138D is sufficient to disrupt dimerization while...

...by a ‘CAC’ codon that is converted to a ‘TAC’ or ‘TAT’ after a C-to-T base editing event. This edit changes...Base editors create specific point mutations in the genome, but they’re inefficient compared to CRISPR... is a bicistronic construct that consists of a mutated mCherry and a downstream constitutively active ...the eGFP reporters the Harris lab individually mutated three codons in eGFP to eliminate fluorescence....eGFP reporters rely on the correction of point mutations in eGFP that results in the restoration of fluorescence...GFP construct, that contains an ACG start codon mutation, and integrated it into human and mouse cells ...events in vivo. As GO relies on the correction of a mutated start codon to initiate protein expression, it ...