.... At Addgene, we use blastn to identify any discrepancies in Sanger sequences, such as mismatches, deletions...determine the potential effect of any nucleotide discrepancies. The blastp and blastx programs are optimized...matrix BLOSUM62, but you may want to check the description of the other matrices to see if another would...plasmids instead! All BLAST images are modified screen shots from the NCBI BLAST website. Additional Resources...

...type of interest. Repeat rounds of infection and screening by PCR enrich for capsid variants with increased...of Chan et al, was neurons and astrocytes. Why screen for IV deliverable AAVs? Typically AAVs are directly...brain. Delivering the CREATE AAV library via IV screens for capsids that are able to cross this barrier...AAV as a tool for gene expression in mammals. Subscribe to Viral Vector Blog Posts Additional Resources...

...C-to-U and A-to-I transcriptome-wide off target effects observed in whole-transcriptome RNA-sequencing, ...amount of off-target editing. In one direct transcriptome-wide comparison, REPAIRv2 reduced off-target...the RESCUE system’s ability to edit endogenous transcripts finding up to 42% editing efficiency. They note...

...Cas9 expression     The requirement for Cas9 transcription and translation delays editing What are Cas9...from Alex Schier’s Lab. gRNAs can be in vitro transcribed from ssDNA, which can be generated by commercial...targeted approach using RNPs has recently been described in vitro, with the hope of eventually using this...PMC5681539. Additional resources on the Addgene Blog Subscribe to CRISPR blog posts Learn about other CRISPR ...

...sequenced genome also permits “whole-genome/transcriptome” studies, in which, for example, the activity...two revolutionary large-scale random mutagenesis screens produced ~1,500 zebrafish mutants that are still...deactivation tools for zebrafish Random mutagenesis screens provided a major toehold into understanding how...one-eyed pinhead mutant from the random mutagenesis screens. Microscopy-based lineage tracing can help illuminate...

...policy and recently announced that of the 16,000 manuscripts that had been processed since the declaration...access of this data is to be made at the time of manuscript submission. The rationale includes increasing...some cases, typically consortia involving genomic screens, the data stream is so prolific that raw information...issue by requiring ever more condensed methods descriptions, legends and limiting numbers of figures such...

... editor or adenine base editor) and reverse transcriptase-mediated prime editing technologies have been... through Addgene. In addition to the plasmids described above, Addgene carries several useful CRISPR toolkits...Toolbox for Multiplexed Plant Genome Editing and Transcriptional Regulation. Plant Physiol 169:971–985 . https...CRISPR/Cas9-Targeted Mutagenesis of the ERF Transcription Factor Gene OsERF922. PLoS ONE 11:e0154027 ....

...including easier automation, higher throughput screening, and a lower requirement for the amount of DNA...transgenic animals may choose Southern blotting to screen founder transgenic animals for the inserted transgene...below), restriction mapping may still be used to screen multiple colonies before selecting a smaller subset...prevails. Sequencing whole genomes or profiling transcriptomes (RNAseq) require sequencing of many samples...

...perform in vitro transcription, and use the IVT product as a template for reverse transcription to create your...process of mouse model generation simpler, as described in an earlier post on generating CRISPR mouse ...'d use ivTRT to create the ssDNA construct as described in Figure 2.  You can also use Addgene plasmids...

...these cases, screening multiple bacterial colonies using one of the detection methods described above can...confirmations like linear and nicked plasmids, as described in this article.   Long-read sequencing Addgene...by growing seed stocks at 30°C compared to 42°C. Screen multiple colonies to isolate the monomer Sometimes...

...inserted in this LTR so you don’t need Tat to transcribe the viral genome in producing cells. Currently...process is to test several protocols that have been described (protocol 1, protocol 2, protocol 3) to find the...transduced by a GFP encoded virus, and the reverse transcriptase activity assay uses qPCR to quantify viral RNA... RNA. All of these methods have been described in a previous Addgene blog Once you know your viral titre...

... to screen for positive clones in most cases, and restriction enzyme digest can be used to screen plasmids...BAC DNA can be modified with any of the below described approaches.    Bacterial strain with integrated...recombination than dsDNA substrates, so you should screen more clones when using dsDNA. Have you used the...

... been described in this blog. If your data does not meet the assumptions for the tests described above...to not have any effect on each other, they have discrete effects on the dependent variable, and those variables...error rate than the multiple comparison tests described above. Violated assumptions and statistics Statistical...

...update, we have added new sections to share and describe some incredible new CRISPR technologies developed...much-anticipated prime editing section and additions describing CRISPR’s ability to make large genome edits using...transposases or the Cascade-Cas3 system. We also describe newly engineered Cas9s with more flexible requirements...

...levels to generate anything from individual transcription units (TUs) used to express your favorite gene-of-interest...genetic circuits with multiplexed guides, mini screening libraries, direct edit optimization, and more....can be invaluable for more complex editing and screening experiments in organisms that don’t yet have these...are designed for flexibility. The Fragmid kit described above works in both mammalian systems and Drosophila...

...researchers at Japan’s Hiroshima University who described their new tool in Scientific Reports in June. ... gap. As Sakuma and colleague Takashi Yamamoto describe in their recent study, the construction system...CRISPR Libraries Enable Genome-Scale, Knockout Screening ...

... The new search bar.   Pro tip! You can use a manuscript title to search for plasmids from a specific ...quickly navigate around the Addgene website without scrolling up and down the page.  Figure 3: The Education... Tools menu Further down What happens if you do scroll down? You’ll find Blugene, as well as information...

...characteristic radiance for some time – there are records describing them dating back to the first century A.D. – ...glowing protein could be used to measure gene transcription, as well as to track protein localization. And...mechanistic understanding of GFP’s function. His lab described the protein’s structure and also discovered many...

...ability to direct enzymes – from nucleases to transcription factors – to specific sequences of DNA has become...proteins The tool she and her colleagues led by Zhao described and demonstrated in a report published in The ...of designer PUF proteins for specific post-transcriptional regulation of endogenous RNA. J Biol Eng. 2014...

..., P1 phage. 2) The post describes an ingenious but simple technique to screen for proteins that could ...Jessica Sacher from the University of Alberta and describes a technique you can use to find phage proteins...