...exchanged contact with Amy C., Addgene’s intern coordinator shortly after hearing her talk about Addgene’...program, teaching me the basics of physics and sustainable design while allowing me to do my internship ...results every week always came with surprises. Originally we thought during the first three weeks that ...capstone project report and presentation, during the final week at Addgene. This report shared my work by breaking...each day, my background, observations, etc. On my final day (today!), I presented my research to Addgenies...

...accessibility of CRISPR-based phenotypic screen datasets. Original published CRISPR screen data is curated and deposited... more than 45 publications, beginning with the seminal research articles that demonstrated the feasibility... Each screen curated in ORCS is linked to the original publication via its PubMed identifier, which can...metadata.   Screen results are reported as the original quantitative gene-level scores provided by authors...rather than sgRNA-level sequence read scores. The original supplemental data tables available with the publication...

...In our case, we will add TAAGCA, resulting in a final Forward Primer sequence of 5'-TAAGCAGAATTCATGTGGCATATCTCGAAGTAC...amplification. We can start similarly, taking the final 18 bases of the ORF, including the stop codon (5...TGGCATATCTCGAAGTACTGAGCGGCCGCTAAGCA-3’) into this calculator we get a final Reverse Primer sequence of 5’-TGCTTAGCGGCCGCTCAGTACTTCGAGATATGCCA...restriction cloning, it is essential that you verify your final plasmid by sanger sequencing. DNA replication by... you use, it is important that you sequence the final product. Additional Resources on the Addgene Blog...

... pathway disrupt target gene sequences (thus inactivating genes), while breaks repaired by homology directed...the biological cause of this repair bias is a fascinating (and yet unanswered) question. Our recent paper...identical dissociation rates for catalytically inactive dCas9 molecules. We followed these experiments...vitro results also demonstrated that catalytically inactive dCas9 produced a “bubble” structure in uncut target...homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA." Nature...

... PMID: 30678704. Sparse cell labeling using recombinase competition Article contributed by Aliyah Weinstein...Weinstein Sparse Predictive Activity through Recombinase Competition (SPARC) is a fully genetic tool for...organism. The SPARC system uses competitive recombination sites to selectively remove a stop cassette ...effector is expressed. By truncating one of the recombination sites in the construct to different lengths,...monitoring in vivo. To overcome these technical issues, Sabrina Büttner’s lab designed a calcineurin transcriptional...

...by Klaus Hahn's lab at the University of North Carolina (plasmids at Addgene). It consists of two domains...and as a consequence, EB1 protein function is inactivated by the separation of its synergistic domains....and colleagues started with the FP mNeptune684 (originally described by Li ZY, et al. PLoS One, 2016) and... editors that utilize dCas9 fused to cytidine deaminases like APOBEC. To achieve this they created a lentiviral...efficient than the BE3 plasmid containing rat APOBEC1. Finally, they show that the ACE reporter can also quantify...

... the protein with enterokinase, which recognizes the 5 amino acids on the C terminal of the tag. Of the...background can be a huge challenge. Epitope tags eliminate the need for target-specific antibodies and have...genomic locus or more commonly via cloning of recombinant proteins. Why use a tag? If you are fortunate...protein that can also be used as a tag! GFP was originally derived from jellyfish (Aequorea victoria) and...

...conversion is a unique characteristic of recombinant antibodies. Recombinant antibodies are plasmid-based and ...The resulting chimeric antibody would have the original variable regions from the parental rat antibody...antibody retains the binding specificity to the original target and can be used with secondary antibodies...is routinely used to increase the utility of recombinant antibody tools, the resulting chimera may have...

... your protein of interest will only occur in combination with that reporter, you can infer its presence...using a mix of different antibody-fluorophore combinations for different markers of interest, you can separately...for distinguishing live from dead cells. The combination of antibody-fluorophores, fluorescent reporters...BV421 and RB545. Fluorophores can also come in combination of two conjugated single fluorophores, like APC-Cy7...be emitted by either of the two. As a result, inaccuracies in the detection of fluorophores (and the markers...should try to avoid or reduce it whenever possible. Finally, we designed a simple panel to analyze transduced...

...you to quickly associate your plasmids with the original publication where you described them. Search for...This publication will be listed at the top of the final plasmid pages and scientists will be asked to cite...repetitive sequences (which can be prone to recombination in bacteria) such as lentiviral, retroviral,...Verification On this page you will see a preview of the final plasmid page that will appear on Addgene’s website...available to the research community. This article was originally written by Tyler Ford in January 2016. It was...was updated by Angela Holmes and Christina Mork in February of 2024.  Additional Inside Addgene Content...

...goals require methods to directly activate and inactivate biological processes. Chemical effectors are ...two-component systems (TCSs). TCSs consist of a histidine kinase that phosphorylates a response regulator (RR). ...from cyanobacteria to a common E. coli histidine kinase. This hybrid construct senses light, and an obligate... two-component system (TCS). A hybrid histidine kinase/photosensor senses various light inputs, and an...issue; some promoter activity persisted in the inactive state. The dynamic range, defined as the difference...

..., the problem comes down to inadequate information about what the original researchers actually did. Admittedly... have to be this way. We can never completely eliminate the risk that a minor variation in experimental...put down a basic outline and refer back to the original paper that everyone in your lab has been referencing...President of Editorial at Cell Press.  She trained originally in cell biology, completing her PhD at the MRC...

...atypicalities, including a patient with amusia - the inability to perceive music as anything but noise. As a ...pop psychology, Musicophilia is accessible and fascinating, all without compromising on the relevant science... Tsang, Science Communications and Marketing Coordinator Non-science favorites  The Mistborn series by...even more sinister. 1000 pages long. Incredibly imaginative, funny, bittersweet, and shocking at times."     ... PhD, majored in Biochemistry or International Finance. A book club could lead to smart, sometimes contentious...

...demonstrated in different cellular environments such as retina or glioblastoma). They also found that exo-AAVs... protects them from antibody recognition and inactivation (György et al., 2014), but scientists have yet...deafness (György et al., 2017), exo-AAV2 in mouse retina after intravitreal injection to transduce bipolar...difficult to reach, and are more resistant to inactivation through anti-AAV antibodies. References György...vector mediates robust gene delivery into the murine retina upon intravitreal injection." Scientific reports...

...so it’s better to start over if that happens.  Finally, if you’re seeing a weird issue and can’t figure...ladder compatible with your imaging system; or a combination of the two. There are many commercially available...SDS for your proteins to migrate…so don’t try eliminating one component entirely. If you are trying to ...bit to find a membrane, transfer, and buffer combination that works. Background If you’re seeing odd background...manufacturer. You can also consider using monoclonal or recombinant antibodies, which have much lower lot-to-lot ...point. You can also consider using polyclonal or recombinant multiclonal antibodies to increase overall signal...enough antibody binding sites Polyclonal or recombinant multiclonal antibodies Protein not properly...

...their target protein is altered by blue light illumination. This ability to reversibly bind and release...tagged, which limits these approaches to use with recombinant proteins. Purifying an untagged protein with ...initiates the extracellular signaling-regulated kinase (ERK) signaling cascade. This signaling is a lot...signaling by using a fluorescent reporter for Erk kinase translocation. These results demonstrate that OptoNBs...pathway. What’s next for OptoNBs? While OptoNBs illuminate a path to light-controlled protein binding, they...

... the synthetic biology scene in 1989 through a seminal paper from Peter Schultz’s lab describing a technique...same for all three components, meaning that the final protocol may involve a sensitive balance of trade-offs...GCE4All is a great place to start. They host monthly webinars, workshops, and a bulletin board on their website...their first protocol is available now. It is for recombinant expression of phosphoserine-including proteins...Site-specific Incorporation of Phosphoserine into Recombinant Proteins in Escherichia coli. 12(21). https:/...

...expression and largely defines the amount of recombinant protein that will be produced. Many common promoters...Positive inducible In the OFF state, the promoter is inactive because the activator protein cannot bind. After...Negative inducible In the OFF state, the promoter is inactive because a bound repressor protein actively prevents...tetracycline-controlled transactivator) is normally inactive and cannot bind the tetracycline response elements...a reporter gene. When light is present, YFI is inactive, preventing repressor cI synthesis and allowing...

...washing and then incubate with Proteinase K to reverse the crosslinking. Finally, purify your DNA with a PCR...fragment size and (2)  incubate with RNAse and Proteinase K to purify the DNA and then determine concentration...isolated your antibody:protein:DNA complexes and finally (5) isolated the DNA from your antibody:protein... on Addgene.org Find plasmids encoding recombinant antibodies at Addgene ...

...any type of DNA or RNA purification to avoid contamination, take extra care when performing RNA extraction...using DEPC-treated water, autoclave the water to inactivate the DEPC.  Ensure that plasticware or glassware... baked at 180°C for at least 4 hours.   If your final RNA sample(s) are resuspended in water or TE buffer...homogenization solution and phenol addition of the original no-kit protocol into one step. After homogenization...resuspended RNA sample will help ensure DNA contamination is not an issue.  References                 ...