... the mutagenesis reaction. Alternative approaches to site directed mutagenesis Point-mutagenesis is fairly...relevant in this age of CRISPR; site-directed mutagenesis generally applies to plasmids, but may also facilitate...modifications.   Site-directed mutagenesis of Plasmids. A) Generation of mutagenized plasmids. PCR primers...also generates 4 bp overhangs. You can then use the Klenow fragment of DNA polymerase I to generate blunt...Series Use CRISPR to Generate Specific Genomic Mutations Resources on Addgene.org Browse Our Molecular...Laursen from Cornell University. Site directed mutagenesis is a highly versatile technique that can be used...analysis of proteins (to perform alanine scanning mutagenesis or targeted substitution of key residues), and...

...work with fluorescent proteins, synthetic biology, gene cloning and expression, as well as guide pages for...plasmids made in your lab, see www.addgene.org/deposit or email deposit@addgene.org for more information or ...Addgene will be at the American Society for Microbiology Microbe meeting here in Boston next week - be... and say hello! Just in time for this meeting, Addgene is launching a new resource page for the Microbiology...organisms we should include by emailing us at help@addgene.org. If you are looking for plasmids used in E. .... coli and yeast, you’re in luck! Addgene has so many of these resources that we created filters just ...microbial technology, sign up to write about it on the Addgene blog. ...

... caters to optogenetics users and adjusts the lasers appropriately (tuned for optogenetics). These lasers...answer a research question. The flip side of the optogenetics coin is materials science-based. Light is delivered...optics is probably the least difficult portion of optogenetics (though it does require a significant amount...become go-to providers of materials needed for optogenetics. There are several important considerations ...with fabrication of the fiber optics needed for optogenetics. First, the length of the fiber optic for the...laser output), 5ms “on”, 50ms “off”.  A pulse generator such as the A.M.P.I Master-9 Pulse Stimulator,...manipulation of the laser power source itself. Pulse generation (TTL pulse) is a standard electronics protocol...

...or negative selection). Next-generation sequencing (NGS) to identify genes of interest. Figure ...target genes for drug discovery or to understand the intricacies of molecular pathways. These screens have ...the targeting of hundreds to tens of thousands of genes with minimal effort. The versatility of Cas effectors.... (2022). AAV vectors: The Rubik’s cube of human gene therapy. Molecular Therapy, 30(12), 3515–3541. https... Adeno-associated virus as a delivery vector for gene therapy of human diseases. Signal Transduction and...Forward genetics screens are a valuable part of the molecular biology toolbox to identify new target ...lentiviral-based CRISPR libraries available at Addgene. The large packaging capacity and stable expression...

...provide an increased understanding of DNA binding and gene regulation. For additional information on CRISPR-mediated...technology using recombinant CRISPR ribonucleoproteins." Genes to Cells 21.4 (2016): 370-377. PubMed PMID: 26848818...interactions between genomic regions by enCh IP‐Seq." Genes to Cells22.6 (2017): 506-520. PubMed PMID: 28474362...the 3x FLAG‐dC as9 protein for enCh IP analysis." Genes to cells 23.4 (2018): 318-325. PubMed PMID: 29480524...RNA-guided platform for sequence-specific control of gene expression." Cell 152.5 (2013): 1173-1183. PubMed...Fujita et al., 2018). Furthermore, the Fujii Lab generated a transgenic mouse line and retroviral expression...specific gRNA-dCas9 complex. When combined with next-generation sequencing, this method allows for the recovery...

...Additional resources on the Addgene blog Find out what makes AAV a versatile tool for gene expression in mammals...provides the AAV replication (rep) and capsid (cap) genes. AAV replication uses the host’s polymerase, but... the pHelper plasmid which expresses adenovirus genes which help mediate AAV viral replication; and 3)...a higher number of viral particles for efficient gene transfer. I figure it’s better to have a bit extra...derived inverted terminal repeat sequences. Human gene therapy methods, 23 1, 18-28. PubMed PMID: 22428977...Resources on Addgene.org Learn how Addgene produces AAV and lentivirus Check out Addgene's virus protocols...3) the transfer plasmid which encodes a transgene of interest that’s packaged into the virus. Two to five...

...insertions, deletions, and point mutagenesis to complex assembly of multiple genes. Primer design for each type...amplified, restriction digested or even a synthesized gene. PCR is the primary method to produce the linear...included in a single PCR, for example for subcloning genes from different templates. All PCRs are run as an... 5’ end of either primer. Subcloning - To move a gene from one vector to another simply amplify the vector...protein-coding sequence, or a mutation can be introduced in a gene, while adding a GFP-tag (Figure 3). Achieving multiple... to the 5’ end of the reverse primer. Mutagenesis - Mutagenesis is performed by designing primers astride...using the same three step protocol:  Fragment generation - Any linear DNA fragments can be assembled in...

...-based genome engineering has revolutionized the gene editing field by making experimental workflows considerably... tools. As a consequence, a critical step in the gene editing workflow - analyzing the data - is often...likely to result in a functional KO of the targeted gene.   Analyze Complex Edits   Traditional Sanger sequencing-based...result in a functional Knockout (KO) of the targeted gene. The analysis can be sorted by any of the parameters...validation tool, and guaranteed editing with the CRISPR Gene Knockout Kit. Please contact us at [email protected]... more efficient than previous methods. Still, generating reliable results from CRISPR edit data requires...detect or analyze complex edits, such as those generated by multiplexed delivery of multiple sgRNAs. Since...

...Addgene is very excited to share the very first edition of our Antibodies 101 eBook! The latest addition... always, don’t hesitate to reach out to help@addgene.org with any thoughts, questions, or feedback. Happy...

...media, you can find us on Bluesky: @addgene.bsky.social; or @addgene on Instagram, TikTok, Facebook, and...we will not delete @addgene on X. This will additionally ensure that the @addgene handle is always associated...an official Addgene account. You can reach out to us directly via email at help@addgene.org, and we invite...Addgene is announcing we have chosen to stop all activity on X, formerly Twitter. If you are interested...mainstay of conversation in the scientific community. Addgene has been considering a gradual move off X as a ...to paid content regardless of user preference. Addgene is a values-driven nonprofit. Recent remarks and...posting, monitoring, and resharing content on X.  Addgene deeply values our community and recognizes the ...