... 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...

... discusses some of the programs SITN runs and describes some of the more popular topics from the Science...telling. Chapter 5: The SITN Blog - Vini and Amy describe how the SITN blog is a source of scientific information...underwater? Chapter 8: Work-Life Balance - Vini describes how she balances working in the lab and working...

... paired DSBs. This method allows for deletion screening by conventional PCR. Biallelically disrupted cells...transfected cells via FACS, primer validation, and screening by PCR for biallelic deletion clones:    ...video? The Orkin and Bauer lab's related paper describes their methods for CRISPR design, cloning, and ...

...These tagging vectors are based on previously described pFA6a-link vectors and include a Kan, SpHIS5, ...former lab at UNC Chapel Hill. Bähler et al.(2) describe a modular collection of plasmids for a wide variety...3xHA, 13xMyc, GST, or GFP). Longtine et al.(3) describe a complimentary set of plasmids for use in S. ...

...process When I first saw the comprehensive job description for this co-op, I was immediately interested ...had stood out to me because it gave me a full description of the tasks I would be responsible for, in comparison...and Geneious, I was able to determine if any discrepancies between what we sequenced in house and the submitted...

...certainly taken notice! Our newest page in the Transcriptional Regulation category already has five blue flame...with eSpCas9, SpCas9-HF1, & HypaCas9 Day 10: Transcriptional regulation: Truncated gRNAs for Regulating ...Chavez) Day 11: Pooled libraries: Genome-Wide Screening Using CRISPR/Cas9 and Plasmids 101: NGS Quality...

...positive selection, negative selection, and/or screening. We’re focusing on positive and negative selection...in this blog post, but don’t worry, we’ll cover screens in a future post. Positive selection: Cells that...with or without your inserted DNA) and further screening will help you determine if the plasmid backbone...