We narrowed to 457 results for: HA tag

...booklet. What has been the most surprising thing about becoming a PI? Tom Ellis has a one word answer...prepared me for what a PI without a personal assistant has to deal with. My inbox currently stands at about ... build a strong, collaborative team. And take advantage of the many resources available from your institution...

...Find Empty Backbones at Addgene Each approach has advantages and drawbacks, which are summarized in the ...for the presence of an insert. Traditionally this has been done with restriction enzyme digest; however...insert-specific primers but also tell you whether the insert has the correct directionality. Verifying the insert ...and are more likely to work in a PCR reaction that has bacterial debris. Use a positive control. A good...

... population level, but each individual cell only has a maximum of one gene knocked out. In this genetically...containing up to around 200,000 different gRNAs. It has been suggested that researchers should use ~400X ...than gRNAs in a screen, to ensure that each gRNA has a good chance to contribute to the screen. In the... If you are lucky and find that a druggable gene has a central role in your system, you could majorly ...guest blogger Fredrik Wermeling! Fredrik Wermeling has a research group at the Centrum for Molecular Medicine...Medicine, Solna, Karolinska Institutet, in Sweden. He has a particular interest in using molecular tools to... differentially expressed genes can be the next stage in making sense of such data. These targeted screens...

...restriction endonucleases. A given prokaryote typically has genes encoding one or a few restriction modification.... Using the same example plasmid as above (which has the XbaI site blocked by methylation), the figure...newly synthesized product from a site-directed mutagenesis experiment? The answer to both questions is the...to methylation if the enzyme's recognition site (TCTAGA) is preceded by GA or followed by TC. As shown ...cleave DNA. DpnI is often used for site directed mutagenesis. During this process, incorporation of a desired...Troubleshoot Your Sequencing Results Use REPLACR Mutagenesis to Mutate Your Plasmids Resources on Addgene.org...

...the opsin you use); the efficacy of optogenetics has already been proven. Therefore, a concrete, well ...scientists who pioneered the development of optogenetics, has genetically manipulated a number of the opsin genes...kinetics of opsin activity/inactivity. Dr. Deisseroth has also generously made many of his optogenetic constructs...stereotaxic surgery (see videos below). The Deisseroth lab has developed many of these AAV constructs with various...promoter of the cre recombinase while still taking advantage of the robust expression via the promoter in the...stereotaxic frame with a digital readout. The advantage is you can “zero” your coordinates at bregma which...engineered to express various opsin genes [7-9]. The advantage of using these lines is, if one is crossed with...

...mammalian systems. Our luciferase collection page has a detailed list of luciferase options. Table 2: Properties...light of a different color instead. This approach has even been used in biosensors where BRET reports on...available. Table 1 compares some general advantages and disadvantages of each approach. Table 1: Properties...levels and improved signal sensitivity. Another advantage: luciferase reporters aren’t susceptible to photobleaching...luminescent substrate. This approach preserves the advantages of luminescent assays (low background, no photobleaching... that produce different colors or have other advantageous properties. This approach can improve live-cell...

...extension PCR. Although each type of cloning has its advantages, most scientists still encounter several ...science.     References: 1. Celie, Patrick HN, Annabel HA Parret, and Anastassis Perrakis. "Recombinant cloning...Plasmid Cloning by PCR Perform Site Directed Mutagenesis by PCR Catch up on Your Plasmid Background with...

...partnership was fruitful and a first version of the study has just been uploaded to the preprint repository BioRxiv... platform; while they are a minority, their help has been essential to push certain projects. The idea...year on, our biggest lesson running the platform has been the realization that there is a great untapped...Crowdfight is also open to other topics.  In this new stage of the project we are focusing on what we call “...

...method While the hybrid dual vector system has a slight advantage over the overlapping and trans-splicing...Adeno-associated virus (AAV) has many features which make it a great viral vector, but its packaging ... Overlapping  Overview: The overlapping strategy has a region of homology, typically ~400-1400 bp long...Drawbacks:  Generally an overlapping split vector has equal or higher transgene expression when compared...efficiency of transgene expression from split vectors has been inconsistent. A greater understanding of basic...

... editors be close at hand? Once again, this post has a lot of acronyms, so we've included a glossary at...or C substrates. The HuidaGene Therapeutics team has deposited their editors, called gTBE and gCBE, with... abasic site. The same protein (or its homologs) has more than one name. Indel Insertion/deletion A ...this promising start, the team used rational mutagenesis to produce the adenine transversion editor AYBEv3...and ABEs, they could be useful for saturation mutagenesis, and they provide a strong proof of concept for...empty space. Over several rounds of rational mutagenesis, the team progressed to gGBEv6.3, which was capable...

...in vivo scientists need a protease reporter that has a large dynamic range and high brightness. The Shu...in the midgut of Drosophila, an observation that has been difficult until now (Zhang et al., 2019). Conclusion...Conclusion FlipGFP is a novel fluorogenic reporter that has a large dynamic range and high brightness. This reporter...minutes (Cabantous et al., 2013). The Shu Lab took advantage of this and came up with a system where fluorescence...

...-homologue recombination was released. This blog has not been updated in response to this paper. Any hint... hint of CRISPR editing in human embryos has been met with a storm of media coverage. But the paper published... the edited embryos proceeded to the blastocyst stage at similar rates to untreated controls. Ma et al...results in fewer eggs. However, the potentially staggering costs of such a treatment on top of the expense...limited their whole genome sequencing to a small percentage of edited embryos. Since the embryos produced...had few predicted off-target effects, but this advantage likely won’t transfer to all genes we’d like to...worthwhile for a given disease if it increased the percentage of disease allele-free embryos to only 60%? Unfortunately...

...prime editing By Emily P. Bentley The David Liu Lab has made two recent deposits focused on optimizing prime...collaboration with the Vallabh/Minikel Lab, the Weissman Lab has developed a new epigenetic editor, Coupled Histone...subtypes, and a familiar challenge for anyone who has done histology on brain tissue is identifying the...magnetic beads through an assembly of biotinylated SpyTag-SpyCatcher linkers and spacer proteins ending with... single alpha helix) and target capture module (SpyTag-GFP nanobody) assembled on beads. C) Micrograph...methods such as chemical crosslinking or tilted-stage imaging. A truncated form of a tardigrade LEA, RvLEAMshort...Release of Methyltransferase (CHARM), with several advantages over the earlier system.  Rather than overexpressing...

...mouse embryos as Cas9 from S. pyogenes and has the advantage of being smaller (Zhang et al., 2016). For...well if you’ve been following the Addgene blog, it has been repurposed for genome editing in eukaryotes,...than that, be nice to your microinjectionist, who has the strategically important job of delivering the...replacement of the murine sequence with a human gene. Advantages of using CRISPR for mouse genome engineering ...the efficiency discussed above extends 4 major advantages of CRISPR mouse editing when compared to more...Post for Tips on CRISPR Gene Editing in Mice Disadvantages of using CRISPR for generating mouse models ...

...within the human genome. Another variant, xCas9 3.7 has 7 mutations found in the REC2, REC3, and PAM interacting...CRISPR toolbox The isolation of novel CRISPR proteins has and will continue to dramatically increase the number...additional targeting flexibility. Cas13a (formerly C2c2) has been adapted for targeted RNA cleavage in mammalian... MH, Tang W, Chen L, Sun N, Zeina CM, Gao X, Rees HA, Lin Z, Liu DR (2018) Evolved Cas9 variants with ...create a specific RNA editor. Cas13 enzymes are advantageous because they do not require a PAM, and RNA targeting...

...as a scientist has been building your creativity all along. In my own career, my team has used these techniques...and instead building an environment where everyone has their ideas heard.  So how can we assure that all...continue advocating for your idea. Our product team has implemented a policy of never rejecting an idea simply...plaguing the scientific community is supply chain shortages. When flasks, conical tubes or pipette tips are...

... and allows targeting of specific cell types, it has a limited capacity to carry large DNA cargos. As ..., the search for smaller, compact genome editors has become a key focus in recent years. TnpB nucleases...nuclease domains (HNH and RuvC-like), and Cas12a has one (RuvC-like), TnpB also operates with a single...21 base pairs downstream of the TAM, creating staggered double-strand breaks like those created by Cas12a... like Cas9. This is where TnpB offers a clear advantage. At approximately one-third the size of Cas9 or...

... - typically within 2-4 months. After the animal has the desired level of antibodies in its bloodstream...their batch to batch variability. Once an animal has undergone a terminal bleed to acquire all of its ...cross-reactivity. When to use polyclonal antibodies One advantage of polyclonal antibodies is that they are inexpensive...background staining (see next section), it may be advantageous if you are trying to detect a protein in low...research, monoclonal antibodies Learn about the advantages of using recombinant plasmid-based antibodies...

...Further, Sanger sequencing has a lower detection limit of 50-20% (although this has been improved in some ...Joung 2016 with permission. Even though progress has been made with off-target prediction algorithms, ...Technique Sequence Information Detection Limit Advantages Disadvantages Mismatch Cleavage Assay Not Provided... Technique Detection Limit Application Advantages Disadvantages GUIDE-Seq 0.1% Cell-based  Searches ..., Yanfang, et al. "High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells...

...CombiGEM-CRISPR: The marriage of two simple platforms CRISPR has revolutionized how we decode the genome by making...guide RNAs (gRNAs) for genome editing in large-scale has led to the rapid generation of genome-wide gRNA libraries...the users’ specific research questions. CombiGEM has been successfully applied to functionally characterize...; Reverse oligo: 5’- AATTGXXXXXXXXGAATAGGTCTTCTCGAAGACCCAAACNNNNNNNNNNNNNNNNNNNC -3’, where NNN and XXX...