...removing 1-20 nucleotides from the cleavage site specified by the gDNA. Depending on your application, this...About NgAgo User's First Impressions Here References 1. Gao, Feng, Xiao Z. Shen, Feng Jiang, Yongqiang Wu...template is supplied. Find Plasmids from the NgAgo Publication at Addgene Cas9 and NgAgo head to head NgAgo ...differences in cleavage efficiency. If this finding is replicated in larger studies, it will represent a key advantage...PubMed PMID: 27136078. Find plasmids from this publication at Addgene. 2. Swarts, Daan C., et al.  “DNA-... PMCID: PMC4697943. Find plasmids from this publication at Addgene. 3. Swarts, Daan C., et al. “Argonaute...

...organoid model (Sun, et al. 2021).  Figure 1: Example schematic from the Organoid EasyTag system...without altering that of the internal control (Fig 1). Because this method relies on the fluorescence ratio...be used to generate organoid cultures with a modification of interest. While the authors optimized the...mechanisms related to morphogenesis, cell fate specification, and human disease.     Sun, et al. bioRxiv...protoplasts, the enzymatic biosensor retains its catalytic activity.  As SLs are also known to trigger seed...parasitization of cereal crops. As a result, agricultural applications of these novel biosensors could have a major...

...B. Gurumurthy, and Masato Ohtsuka. Nat Protoc. 13(1) (2018):195-215. PubMed PMID: 29266098 Creating knock-in..., with protocols using dsDNA templates achieving ~1-10% efficiency. Easi-CRISPR uses a long ssDNA template...the gRNA design process and rules for various applications is necessary going forward. The standardization... I think I may have finally grasped just how complicated DNA repair pathways are. I fully admit to being...Alt-NHEJ), has been harnessed for CRISPR knock-in applications. CRISPR 101: Targeting RNA with Cas13a (C2c2...for CRISPR Genome Editing Other resources and publications CRISPOR gRNA design tool Researchers at the ...

...target DNA quickly and accurately.  Figure 1: Steps in PCR reaction   This process of denaturation...extension is repeated 25-35 times to exponentially replicate the target DNA of interest. This entire reaction...have been developed for different scientific applications. These PCR methods all use the same basic PCR...the final cycles which would slow or stop PCR amplification. Other times, the PCR reaction may not be 100%...quantified by qPCR. qPCR is used in a variety of applications including gene expression profiling, studying...concentration, unlike qPCR where results can vary across replicates. ddPCR can be used to quantify DNA sequences ...droplet contains the material required for PCR amplification. Following the PCR reactions each droplet is...

...antisense RNAs inhibit plasmid replication.   Figure 1: The replication problem. Iterons are repeated...actually does, and neither plasmid is maintained (Figure 1).  The partitioning problem Plasmid incompatibility...Agrobacterium tumefaciens. Genes & Genetic Systems 77:1–9 . https://doi.org/10.1266/ggs.77.1 Pubmed PMID: ...Origin of Replication? See our Plasmids 101: Origin of Replication blog).  The replication problem Plasmids...directly inhibit replication by binding to the origin of replication and blocking replication machinery (del... of microbial plasmids: classification and identification based on replication and transfer systems and...plasmid that contains the origin of replication and the replication control machinery (Need a refresher...

...with a single insertion because it will segregate 3:1 in the T2 generation. You also have no control over... transgenic plants in the process.     References 1. Emami, S., Yee, M. and Dinneny, J. R. (2013) ‘A robust...transformation’, Plant Methods. BioMed Central, 2(1), p. 19. doi: 10.1186/1746-4811-2-19. PubMed PMID:...Agrobacterium tDNA insertion likely precipitated domestication of sweet potatoes (Kyndt et al., 2015)! It sounds...

...By the end of 2023, our catalog had grown to 879 viral catalog items (Figure 1). The service caught on...over 100,000 viral vector preps!   Figure 1: A) Cumulative AAV preps available in the Addgene ...Neuromethods, 195, 77–101. https://doi.org/10.1007/978-1-0716-2918-5_5 Haery, L., Deverman, B. E., Matho, K...repository. GCaMP, a calcium indicator (biosensor, by Addgene’s categorization) was first developed in 2001....   Figure 2: Numbers of AAV tools, by category, available from Addgene as of 2023. A large... calcium and voltage sensors, are the broadest category of viral vector tools, which may help explain ...number of requests for vectors in the “controls” category, which includes things like viral vectors that...

...level of expression needed for most applications.   Figure 1: With blue light, the VVD domains of... that dimerizes upon exposure to blue light (Fig. 1). Another blue light-inducible dimerization domain...in @nchembio Title: Bye bye L-arabinose drive. Dedicated to Edo, Armin and @KhammashLab. I hope you all...

...following three strategies might help!     Figure 1: Species on species staining can be a problem because...same host species as your secondary antibody since 1) your secondary should not recognize Fab fragments... “species on species”) immunochemistry (IHC) applications an issue? The culprits are endogenous IgGs in...Partridge TA (1998). A New Blocking Method for Application of Murine Monoclonal Antibody to Mouse Tissue...

...determined LY6A, also known as stem cell antigen-1 (SCA-1), is the receptor used by PHP.B to reach the CNS...William L. Stanford. "Concise review: stem cell antigen‐1: expression, function, and enigma." Stem cells 25.6...Mbp region of the mouse chromosome. Of the genes located in this stretch of DNA, Ly6a seemed the most likely...

...acceptable. Aims should be mechanistic, doable by 1-3 people in 1-5 years, and contribute to resolving the overarching...possible, do at least two full length practices (~1 h) and get feedback. It’s also helpful to practice...anticipated outcome, and payoff for each aim. Learn to communicate your story with only the most salient details...

...guide RNA (sgRNA), provided it meets two conditions:  1) The sequence is unique compared to the rest of the...bystander bases” if you choose this system.  Fig. 1: Cytidine deamination takes place on the free strand...temperature-controlled genome editing. Nat. Commun. 2017 81 8, 1–9 (2017).Walton, R. T., Christie, K. A., Whittaker... than ever to make precise, targeted genome modifications. Cas9 has been modified to enable researchers...instead.  Choosing a Cas9 for site-specific modifications Site-specific deletions and knockouts Some experiments...mutation at a specific site e.g., deleting the catalytic residue of a protein or knocking out a very small...flexibility as to where your sgRNA needs to be located and there may not be a NGG PAM site anywhere to...

...oligodeoxynucleotides)) are commonly used for smaller modifications (~1-50 base pairs). Small edits require as little...Easi-CRISPR was shown to increasing editing efficiency from 1-10% with dsDNA to 25-50% with ssODNs in mouse models...ribonucleoproteins. Quadros RM, et al. Genome Biol. 18(1); (2017). PubMed.  Additional Resources on the Addgene...pathways of DSBs, with a focus on repair for genome modification purposes. How does homology directed repair...specific mutations into the damaged DNA if these modifications exist in the template DNA. There are several... genome engineering world and made targeted modifications feasible and even easy. This targeted-break ...that can be harnessed by researchers for many applications. Your favorite Cas enzyme, a guide RNA to your...

...sgRNA1), 2; darker than control, 1; very dark). Brighter fluorescence indicates a more successful gRNA design...She loves to communicate the latest and greatest in scientific content.      References 1. Cho, Seung ...In vitro screening for the best gRNA Figure 1: Validation of double strand break (DSB) mediated ...plasmid using standard cloning techniques (The XX indicates where your target is inserted, disrupting the ...effective ones to increase your chances of gene modification in vivo. Find Plasmids for Testing Your gRNAs...et al., 2014). The plasmid is sturdier than the delicate RNA and therefore there has less chance of contamination... the validation assay should give an accurate indication of how many pups will contain a mutation and ...

...Full "Applying for a Faculty Position" eBook Here 1. Have a competitive CV You don't necessarily need ...that people would agree is important to understand. (1-2 sentences). For example: Distinguishing correctly... What will be the focus of your first grant? (1-2 sentences) Simply stated, what are your aims... do need multiple first/corresponding author publications in quality journals. It helps to come from the...a job. The currency of scientific success is publications and grants. 2. Write succinct and easy to understand...humbling. My conviction that I had a flair for communication was rapidly dispelled. She would ask me, "Is...succinct. Slide decks leads to the opposite and can suffocate an exciting idea in a swamp of unnecessary details...

...Check out Addgene's New Viral Service! References 1. Hay, R.J., Macy, M.L. & Chen, T.R. “Mycoplasma infection...contaminations.” Methods in Molecular Biology 946 (2013). 1-13. Pubmed PMID: 23179822. 5. Segura, M.M., Kamen,...Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors."Nature protocols 4.4 (2009...positively charged polycation, polybrene. Interestingly, one study found that alternative polycations such as DEAE-dextran... freshly collected lentivirus for downstream applications. While this ensures that the highest titer virus...lentivirus may want to consider testing a variety of cation and serum sources to ensure the best possible transduction...establishes the best conditions for specific applications, stick with them! Changes in such variables ...

...animal will repeatedly nose-poke at the active hole [1, 2].    Place preference/place aversion also models... can be explained primarily in two different ways 1) your hypothesis is wrong or 2) there is some problem...follow him on Twitter @derekpsimonphd.    References 1. Stuber, Garret D., et al. "Excitatory transmission...EthoVision. Consult the literature to find all the applications of optogenetics relevant to your field. Ultimately...Inhibition of neuronal activation is a little more complicated since basal neuronal activity in your brain region...technology is improving and may one day reach the sophistication of mouse transgenics. Everything that has been... and memory in opioid addiction and science communication/science writing. Derek is also a pianist and...

...and is recommended for use in western blots (Figure 1). If you prefer to produce your own antibody or want...are also available from Addgene!   Figure 1: Experimental validation for Anti-V5 [IPI-SV5-Pk1]...biophysical studies of protein degraders. Nat Commun., 15(1), 8885. https://doi.org/10.1038/s41467-024-52871-9...performs well in many different antibody-based applications.  IPI’s recombinant version is a fusion of mouse...with additional cofactors or using significantly truncated forms that limit its functionality. To make biophysical...cellular processes in C. elegans: mScarlet3 for applications needing intense brightness (such as low-expression...luciferase fragment (expressed separately) assemble and catalyze the luminescence reaction (Figure 5). The researchers...

...sequencing. DNA replication by PCR has error rates that range from roughly 1 per 500bp to roughly 1 per 10 million...not cut within your insert Are in the desired location in your recipient plasmid (usually in the Multiple...amplify and design primers that will bind to and replicate it. The following image shows the ends of the ...Forward Primer will use the sequence 5'-ATGTGGCATATCTCGAAGTAC-3' for the region that binds the ORF and...primer, making our Forward Primer 5'-GAATTCATGTGGCATATCTCGAAGTAC-3'. Many restriction enzymes do not cut...final Forward Primer sequence of 5'-TAAGCAGAATTCATGTGGCATATCTCGAAGTAC-3'. For the Reverse Primer, the design...need to use the reverse complement to get PCR amplification. We can start similarly, taking the final 18...

...your budget, you should take the following steps: 1. Define your timeline Start with your thesis in mind...these decisions in the following order of importance: 1. First make decisions based on experimental timelines... several factors aren’t taken into consideration. 1. Always focus on the science When negotiating finances.... After identifying all the major equipment, categorize all of your minor tools, like pipets, or mini-centrifuges...titles in the regions where the host institute is located. For example, salaries in Kansas versus salaries... most start-up P.I.’s. It doesn’t have to be complicated, or difficult to create this list. Start BEFORE...discounts, collaborations, access to internal award applications, etc. If this is the institute that can help...