...The pegRNA consists of a CRISPR gRNA extended to include a primer binding site (PBS) complementary to the...genome and a reverse transcriptase (RT) template including the desired edit. Figure 1: Prime Editing...BioRender.com. RNA protectors epegRNAs: epegRNAs include structured motifs at their 3′ end to protect them...binding site (Nelson et al., 2022). PE7: This editor includes the fused RNA-binding protein La to protect the.... That’s all our tips for today! Each tip includes a citation to the paper it was drawn from. If ...

...These tables are not inclusive of all methods, nor are these methods limited exclusively to in vitro cell ...are required for any application. If you want to include a homology directed repair (HDR) template to create... table below summarizes the various components included with each expression system as well as suitable...components into mammalian cell lines is quite broad and includes several different methods, so we've summarized...

...Gate Modular Cloning (MoClo) Kit. What does it include? Patron: Sainsbury has a mandate to share and educate...things for day-to-day life in a plant lab, so it includes everyone’s favorite promoters, plus a few new ...resources. We wanted to expand the toolkit. We have included a wider range of terminators than are commonly...a modern toolkit for those working in plants, including many things people already use. It makes the switch...Addgene recently to explore their use in plants, including FRET tools, double dimerization GFPs, and circular...

...will discuss later.       Standard curves must be included every time the assay is run, ideally with freshly...curves vary depending on a number of factors, including the user, equipment, assay incubation time, etc...curves must be validated for each run. To validate, include a positive control of known concentration in the...Dynamic range depends on a number of factors, including the dilution series of the standard and the minimum...account the range of concentrations possible.  Include both a standard curve and a positive control in...

...screen for molecular interactions in the cell, including protein-protein, protein-DNA and protein-RNA interactions...reporter gene (3, 4), there are now many variations, including many that use auxotrophic rescue genes as reporters...likelihood of indiscriminate reporter gene activation. Including a prey-only control also provides a baseline level...reporter signals depend on a number of factors, including protein expression and correct folding, post-translational...-hybrid systems available in model organisms, including bacteria, alternative fungi (C. albicans, pC2HB...

... the 1930’s in a range of biological studies, including investigations into environmental pollutants and...organism with a clearly established body plan including head, eyes, and tail. Compared to the opaque mouse...process. Additional zebrafish applications and conclusions Zebrafish are also popular outside of developmental...not ideal for every application. Disadvantages include their duplicated (i.e., redundant) genome, their... these caveats, several features of zebrafish—including the available genetic resources and transparency...

...that went beyond obtaining “omic” data and had to include clear plans to connect Genotype to Phenotype thus... is to ensure that, in general, these labs all include materials sharing in their dissemination plans ... diversity in the social bonds that they form including genetic mechanisms that control who a female wants...of basic evolution of fungi and eukaryotes and includes the fungus that is killing frogs all over the ...that most cephalopods die after they lay their 1 clutch of eggs? Propagation is thus quite difficult. CRISPR...

...non-functional hedgehog, the denticles actually clump together forming one patch on the surface, giving...contain components of the SB transposon system including those from the original publication, Ivics et ... been studied in many contexts within the fly including oogenesis and cytokinesis. Why name this protein...activate bagpipe which is localized to segmental clusters of the mesoderm. This subdivision of the mesoderm...that contains bagpipe from the Deplancke lab. Conclusion I hope you enjoyed this tour of Addgene’s new...

...multiplexed elements. The team designed sgRNAs that include an internal barcode and separated multiple sgRNAs...was first described in Anzalone et al., 2019, including modifications to the Cas9 or reverse transcriptase...coding sequences for a range of applications, including CRISPR activation by dCas9-VPR, prime editing,...REVeRT in various contexts in vitro and in vivo, including in human retinal organoids and live mice. Importantly..., Parkinson’s, ALS, and Huntington’s Disease, including plasmids, antibodies, viral vector preps, CRISPR...

...interactions. For difficult to release proteins, including those in the nucleus, more stringent buffers may... may be needed. Since many factors, including salt concentration, ionic strength and pH, affect binding...may degrade the target protein. To prevent this, include protease and phosphatase inhibitors in the lysis...Controls As with any experiment, it is critical to include proper positive and negative controls for an IP...sample that does not. Common negative controls include knockout cell lines or tissues that do not express...

...spacers of the crRNA can be customized as well, including concatenations into crRNA arrays targeting multiple...by sacB counterselection.  The MUCICAT toolkit includes versions of plasmids with different induction ...terminators can serve to interrupt genes (or gene clusters) in metabolic engineering. So far, we’ve used ...concentration. Image from the Yang lab.   In conclusion, the MUCICAT system has the following advantages...engineering with improved cargo dedicated to gene (cluster) interruption.     Plasmids for each of these...

...depend on target and sample type. Example steps include: permeabilization, antigen retrieval, and sectioning...available, and your experimental goals. Example steps include: counterstaining, mounting sample on slides, and...couple of steps are preliminary steps that are not exclusive to IF, but they can have a significant impact ...you optimize your protocol, always remember to include controls to show that your antibodies are binding...antibodies validate them in a variety of applications, including IF (or IHC or ICC), and will share some of their...

... blue light CRY2-CIB1 heterodimerizes or forms clusters under blue light Check out our Optogenetics Guide... One great way to do this is to use CRY2-CIB1 clustering. Fuse either CRY2 or CIB1 to your protein of ...Rab protein vesicle trafficking via CRY2-CIB1 clustering. Image adapted from Aljiboury et al., 2023, under...earlier work on Rab11, the team used optogenetic clustering of Rab8, Rab11, and Rab35 to understand their...attractive strategy for engineering other enzymes. Conclusion We hope this post has illuminated some of the...

...editing to change the genetic code of plants, including the development of crops with longer shelf life...to engineer the genome of many plant species, including commonly used model organisms like Arabidopsis... Medicago truncatula and several crop species including potato, corn, tomato, wheat, mushroom, and rice... used RNA pol II promoters for Cas9 expression include the ubiquitously expressing cauliflower mosaic ...toolkits for creating plant expression plasmids, including plant CRISPR plasmids from the Yiping Qi lab and...

...Symptoms of AIDS include rapid weight loss and an increased risk of infection, including both common infections...at increased risk for other chronic diseases, including dementia, gut disorders, nerve damage, and cardiac...could be used to combat this problem, just as ART includes multiple drugs to lower the odds of developing...near the Cas9 cut site, leading Wang et al. to conclude that some Cas9-derived indels may not ablate viral...

...workplace bullying? Threats to professional status – including belittling opinions, public professional humiliation...competence procedures Threats to personal standing – including undermining personal integrity, destructive innuendo..., intimidation, sexual harassment Isolation – including preventing access to opportunities, physical or... withholding necessary information Overwork – including undue pressure, impossible deadlines, unnecessary...unnecessary disruptions Destabilization – including failure to acknowledge good work, allocation of meaningless...

...technology can be used for numerous applications, including transgenesis, gene-trap screens, and gene editing...manipulations in animal models or cells lines, including stem cells, without leaving behind any accessory...piggyBac™ transposon containing a selection marker is included in a standard homology directed repair template... used for a variety of research applications, including making transgenic cell lines and genetically-modified... using a variety of gene editing technologies including Zinc Fingers, TALENs, and CRISPR-Cas9 nucleases...

...Wilson and Doudna, 2013). RNAi consists of two non-exclusive gene silencing pathways: transcriptional and post-transcriptional...afflicted by pervasive off-target effects. This conclusion was based upon numerous phenotype discrepancies...importance of using orthogonal methods to confidently conclude the function of a gene of interest. Tools for ...discussed below). Examples of these constructs include pGAPi, which allows easy creation of long hpRNA...experiment are now available on Addgene. These include the construct that allows for easy insertion of...

...various sources that are difficult to trace. These include the laboratory personnel, the serum, the cell culture...cross-contamination and due to poor lab techniques. These include reusing pipettes for multiple cell lines rather...Contamination! Tissue culture media often have indicators included in the media that turns yellow from lowered pH...the mycoplasma DNA appears in the form of small clusters, apart from the cellular DNA. Fluorescent DNA ... periods and affect the total cell viability. Conclusion To sum up, it is always important to prevent ...

...high as 1-3%. In light of this finding, we now include guidance on the plasmid page of DIO/FLEx AAV vectors...you intend to use performs in your system. This includes a thorough testing of both the serotype and promoter... is reached you may obtain a false negative or conclude the viral vector is not working.  When using a...off-target expression that can confound your results.  Conclusion Using AAV vectors in your experiments requires...system. It is very rare for a methods section to include all of the variables that need to be accounted ...