...s lab at UC Davis is to generate a genetically encoded fluorescent sensor for serotonin. According to ...Addgene.org Find fluorescent protein biosensors Browse Plasmids from the Tsien lab Browse the Michael Davidson...site-saturation mutagenesis, and medium-throughput screening, she increased the affinity of the sensor for serotonin...overwhelming response and were excited to see the many creative uses of fluorescent proteins. We’re happy to announce...thousands of protein variants simultaneously  With increasing sequencing power, scientists are able to quickly... protein-coding pathogenic human variants are decreased in protein abundance, possibly due to misfolding...neuromodulator, and evidence suggests that it is incredibly important for nearly every aspect of life.” Unger...

...Antibodies Resources on Addgene.org Find plasmids encoding recombinant antibodies at Addgene ...could mean needing to increase crosslinking to counteract the effects of increased sample agitation; a ...such as qPCR or next-gen sequencing. ChIP is an incredibly useful application for exploring these interactions...crosslinking, which uses formaldehyde or UV light to create strong but reversible bonds. This will keep the... necessary for most ChIP experiences, but the increased binding can mask the epitopes antibodies recognize...shorter sonication time could be an opportunity to decrease crosslinking. (Homogenization for a tissue sample...

...al., 2007). Brainbow uses a Cre/lox system to label neurons. Genes encoding the fluorescent proteins are...fluorescent protein genes, Cre recombination is required before fluorescence can occur. Cre recombinase can be...developing fish via Cre protein microinjection or crossing of Zebrabow animals to an inducible Cre line. The Schier...gene directly following the promoter is expressed. Cre-mediated deletion of one or several of these genes...system. One such system is Brainbow 2.0, which uses Cre to trigger the inversion of two genes aligned head-to-head...head-to-head. Another variation, Brainbow 2.1, uses Cre to trigger the inversion of two tandem sets of genes...a specific tissue. XFPs are introduced using a plasmid construct called Zebrabow-GateDest containing an...

...until now. Alice Ting’s lab has developed a new, plasmid-based biosensor to visualise changes in histone... Cindy Y. Jao, and Alice Y. Ting. "Genetically encoded fluorescent reporters of histone methylation in...Additional Resources at Addgene Find the Ting Lab plasmids at Addgene Browse More Fluorescent Protein Biosensors...grandsons that decreased their likelihood of death from cardiovascular disease but increased their risk of... DNA methylation patterns, either large increases or decreases. Hypermethylation of CpG islands in the... units of the FRET pair together and causes an increase in the FRET signal. Subsequent demethylation separates...

...and general DNA repair Reduces plasmid recombination, increases plasmid stability recBCD Exonuclease...mutation Improves plasmid yield F Host does (F') or does not (F-) contain the fertility plasmid. A low copy-number...copy-number plasmid, encodes proteins needed for bacterial conjugation. Genes listed on F´ are wild-type...Vectors Looking for More Plasmids 101 Articles?  Find them all at our Plasmids 101 Topic Page!     ...make major improvements such as those that increase plasmid yield and/or DNA quality. Table 1 below outlines...Exonuclease V activity abolished Increased plasmid stability, reduced recombination relA or relA1 Relaxed phenotype...General cloning and storage of common plasmids, blue/white screening. F- endA1 glnV44 thi-1 recA1 relA1 ...

...efficiency of plasmid delivery to a cell. In contrast, TREE provides a read out for plasmid delivery and... histidine at amino acid 66. This histidine is encoded by a ‘CAC’ codon that is converted to a ‘TAC’ or...antibiotic resistance markers, and other enzymes such as Cre-recombinase drastically expanding the base-editing...Addgene.org Browse the CRISPR guide Find CRISPR plasmids for your research ...Base editors create specific point mutations in the genome, but they’re inefficient compared to CRISPR...experiments. In the past few years, scientists have created an array of base editing reporters that can help...Cas9-mediated editing in real time The Harris lab created ACE, a reporter that monitors APOBEC (a family ...

... Addgene’s experience helping scientists share plasmids, especially those of the CRISPR variety (see slides...is in one of the top 10 countries in number of plasmid requests from Addgene. Addgene - Canton Nucleic...transcriptome sequencing methods including work done by the ENCODE project consortium did not do a good job of seeing...resulted in a cell migration defect. They have created a circRNA website so scientists can view public...developments. Using the Tet-on system, his lab has created a tetracycline cas9 inducible knock-out system....

...HDR efficiency with circular plasmid templates is generally low; to increase the frequency of edits, researchers...of a homologous donor (e.g., sister chromatid, plasmid, etc.), and consists of three pathways: classical...disrupted in the donor. Choosing a type of donor: plasmid, oligo, double or single stranded? The size of ...longer than 200 bases can be difficult, thus dsDNA plasmids are generally preferred for large insertions such...researchers have designed self-cleaving plasmids that liberate the targeting region from the vector. Alternatively...these are generally more toxic to cells than their plasmid counterparts. Another consideration for dsDNA templates...., 2017). Easi-CRISPR in vitro transcribes RNA encoding your repair template, then uses reverse transcriptase...

... resources on the Addgene blog Plasmid 101 Protein Expression Plasmid 101 Biotinylation Other resources...vivo studies (Che and Khavari, 2017). Find APEX plasmids at Addgene! Since the development of BioID and... specific locations in the cell.   Find BioID2 plasmids here APEX2: A more sensitive ascorbate peroxidase... They named this mutant APEX2.  Find the APEX2 plasmids! TurboID and minturbo: Rapid and non-toxic proximity...allowing for more precise temporal control.  Find plasmids for TurboID and miniturbo Split-BioID: A conditional...streptavidin and separated via flow cytometry.  Find plasmids for EXCELL here! Since BioID was developed in ...Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy." Nature biotechnology...

...Antibodies Resources on Addgene.org Find plasmids encoding recombinant antibodies at Addgene   ...this approach can greatly increase the sensitivity of an assay by increasing the number of signaling molecules...green; and proteins are represented in orange. Created with BioRender.com.    How do secondary antibodies...shorter than that of the primary antibody, due to increased binding affinity, many indirect antibody protocols...are designed to take place over multiple days. Increased run time is considered one of the major drawbacks...specific isotype subclasses of primary antibodies can increase the possible number of target proteins.   The ...immunohistochemistry advances have found ways to increase signals from conjugated primary antibodies. The...

...realized we could apply the lessons learned from our plasmid lab to develop a reliable and robust high quality...offer many resources for producing vectors from plasmids — including the protocols we use in our lab — ...while viral vectors are not as easy to ship as plasmids, they still have a global reach. From 2016–2023...vectors in our repository are produced from only 413 plasmids containing a genetic payload. While AAV9 and AAV1...having reliable ready-to-use vectors made from plasmids in the Addgene catalog would be broadly useful...simple as it seems. Take, for instance, the GCaMP-encoding viral vectors available in the repository. GCaMP...conditions. Sharing our experiences Addgene’s plasmid repository gave us the experience to launch a high-quality...

...Antibodies Resources on Addgene.org Find plasmids encoding recombinant antibodies at Addgene Topics...basic guidelines below, you’ll likely be able to create a small, usable panel.  First, fluorophores vary...

...both over- and under-expressing clones. If GFP-encoding cDNA is co-transfected at a favorable ratio to...Time CRISPR User) The Challenges of Cell Culture Plasmids 101: Mammalian Vectors Resources on Addgene.org...Our Protocols Pages Browse Fluorescent Protein Plasmids Find Mammalian Expression Vectors ...amount of agarose/DMEM should be proportionately increased). For some cells, add a drop of serum to aid in...up, most will eventually grow within a week. We screen the wells in groups as they become confluent, taking...important to have a functional assay in place to screen your clones. Depending on the protein, we have ...SH-SY5Y cells, as these cells did not seem to form discrete colonies but constantly migrated away from each...

... entire adult brain, using a plasmid that expresses a genetically encoded calcium sensor, GCaMP6 (Nguyen... eBook Read our plasmids 101 blog series Resources on Addgene.org Browse our plasmid collection pages...developing zebrafish pancreas. They first tagged these cells using plasmids. Then using HOTcre, a method that... resistant to chemotherapy. You can find these plasmids at Addgene. Another advantage of mice as model... feeding the worm bacteria expressing specific plasmids (L4440) that contain your gene of interest (Fire...al., 2015). If you are interested in C. elegans plasmids and resources check out Addgene’s Worm Expression...pages Read our molecular biology reference Find plasmids for your research ...

...Improvement of pCVD442, a suicide plasmid for gene allele exchange in bacteria." Plasmid 51.3 (2004): 246-255. PubMed...exogenous enzymes, require multiple transformation and plasmid curing steps, and can generate off-target mutations...a mutated allele introduced from a conjugative plasmid replaces the chromosomal copy. This is a versatile... These two steps are (Figure 1): The initial plasmid integration step (the so-called “single crossover...difficult, we have also included versions of pTOX that encode one of two visible blue or magenta chromoproteins... an allelic exchange experiment? Find the pTOX plasmids here! Continued development of new allelic exchange...Additional resources on the Addgene blog Read our Plasmids 101 blog posts Find our CRISPR blog posts Learn...

... phosphatase activity: In yeast, the PHO8 gene encodes a vacuolar alkaline phosphatase. Normally PHO8 ... Resources on Addgene.org Fluorescent Protein Plasmids and Resources Fluorescent Protein Guide: Biosensors...synthesized at the ER, delivered to the vacuole via the secretory pathway, and then cleaved to generate an active...In both cases, red fluorescence in the vacuole increased with longer exposure to autophagy-inducing conditions...

...that vectorization strategies include creating an AdV transfer plasmid with the E1 and E3 (Early 1 and Early...replication (Harrach, 2014). Given that the early genes encode proteins essential to initiating the whole process.... These genes’ functions are rescued by helper plasmids used during production and/or by genes present...made available by this deletion from the genomic plasmid can be used to house a recombinant sequence (transgene...late genes are usually present in the transfer plasmid for the majority of adenoviral vector systems used...

...Addgene blog:  Plasmids 101: What is a plasmid? Plasmids 101: Origin of ReplicationPlasmids 101: PCR...process known as plasmid multimerization. Plasmid Multimerization Early studies on plasmid topology recorded...ligate the plasmid, and re-transform the ligated plasmid to recover the monomeric form of the plasmid. Have...copy number plasmids causes instability: CoIE1 encodes a determinant essential for plasmid monomerization... monomer of Plasmid 12259: pMD2.G (depicted on the left) is ~5.8 kb in length. The plasmid can exist on... the sequencing results for their plasmids, they found the plasmids were much longer than expected, and...tandem. Such plasmids are known as multimers, and they are the result of multiple plasmid copies combining...

...Turquoise (CeNL; Plasmid #85199), Yellow (YeNL; Plasmid #85201), Orange (OeNL; plasmid #85202) and Red ... Researchers have been sharing plasmids ever since there were plasmids to share. Back when I was in the...specific strategies and tactics for getting the plasmids he wanted. Addgene makes it so much easier to ...kDa. An example is the CRISPaint-NLuc construct (Plasmid #67178) for use in the system outlined in Schmid-Burgk...CyOFP1-NLuc-CyOFP1; Antares). Direct injection of plasmids for Antares or firefly luciferase into mouse livers...Red (ReNL; plasmid #85203) NLuc fusions. The Nagai lab has additionally deposited more than 25 fusions of.... This construct is available through Addgene (Plasmid #83926). Live-Cell Voltage Sensor  Ingaki and ...

...page and can be used to create fusions with a gene of interest. These plasmids will be most useful for...transfection). Future of FRET The first genetically encoded FRET biosensor, Cameleon (Miyawaki et al., 1997...biosensors too, as another laboratory may have already created the sensor that you need. FRET biosensors designed...multiple FRET biosensors simultaneously would further increase the amount of correlated information available...