...KSgenome and seeing the intricacies of DNA repair has emphasized how essential these studies are to improving...alternative non-homologous end joining (Alt-NHEJ), has been harnessed for CRISPR knock-in applications. ...29266098 Creating knock-in mouse models with CRISPR has been challenging, with protocols using dsDNA templates...

...for mapping where a protein binds genome-wide, and has been widely applied in many model organisms and cell...proteins (Figure 1). Importantly, since Protein A has weaker affinity for mouse IgG, it is helpful to incubate...prior to the addition of Protein A MNase. Once MNase has been localized to target sites, the nuclease is briefly...

...Show that as the story has progressed, you or your lab or science or your CV has grown as well. Find your...Task, Action, Result) interview technique, which has been addressed in much better pieces than I’m able...

...require heavy optimization. Claudia Vickers’s lab has designed novel SL fluorescent biosensors by integrating...showed that the receptor-based fluorescent biosensor has a high sensitivity (50 nM-500nM range) and a high...recent months: Our selection of GCaMP8 AAV preps has expanded. Check out the new selection of ready-to-use...

...The UN General Assembly has declared 2024 the International Year of Camelids. The declaration is intended... antibody libraries to generate new sdAbs. There has also been some interesting work done developing transgenic...al., 2021.   The single-domain antibody field has come a long way since the serendipitous discovery...

...channelrhodopsins. HcKCR1 has a maximum absorption of 540 nm and two-photon excitation around 1,040 nm, has larger photocurrents...anti-c-Myc [9E10] (Even, et al., 1985), the Myc tag has become one of the most commonly used protein tags...

...inhibitory GABA-ergic interneurons, Gordon Fishell’s lab has developed a new AAV approach using regulatory elements...fluorescent protein - mScarlet! The Dorus Gadella lab has recently developed a synthetic red fluorescent protein... imaging and in protein fusions. The Gadella lab has deposited all mScarlet constructs with Addgene. ...efficiency Blue flame depositor Yannick Doyon’s lab has developed a new method to increase CRISPR editing...increases the probability that the locus of interest has also been edited! Thus, ouabain selection enriches...coselection are available at Addgene, and the Doyon lab has also provided a detailed protocol at Protocol Exchange...Essential gene Knockdown library. Although B. subtilis has a fully sequenced genome, many applications in biotech...

...! Lentivirus envelope vector pMD2.G This plasmid has been requested nearly 6,000 times (over 800 orders...broad usefulness of pMD2.G and VSV-G. This plasmid has been used in studies of the secretome, ubiquitin ...

...and insert a linear fragment of DNA (insert) that has been cut by compatible restriction enzymes. An enzyme...one found on the left side of the image below. It has a promoter (blue arrow) followed by the restriction...finished plasmid   Once it looks like your ligation has worked, you will need to pick individual bacterial...purpose. Ideally, once you know that your plasmid has an appropriately sized insert, you should send it...plasmid for more details. Once your complete plasmid has been verified, you’re ready to get experimenting!...

...Not the case, as I’ve discovered.) As someone who has now seen both sides of the postdoc experience, I’... lab is closely involved with clinical trials or has ties to industry, the drug discovery process can ...Genentech, will not. The point is that each company has a unique approach, and some might suit your needs...currently a Postdoctoral Scientist at AstraZeneca. She has a background in cell and developmental biology and...

...the growing spectrum of fluorescent proteins (FPs) has been an invaluable resource for studying the organization...sequence (like GFP) can be inserted at the DSB. CRISPR has allowed scientists to tag and light up endogenous..., and T2A-TurboGFP-PEST), and small epitope tags (HA, Myc, Strep tag II, AviTag, HaloTag, SpyTag). The...characteristics. For example, Masato Kanemaki's lab has developed a CRISPR/Cas-based system for tagging endogenous...

... ddPCR.) qPCR Quantitative/real-time PCR (qPCR) has been used for copy number assays for years. qPCR ...and quencher to quantify specific amplicons.  Sybr has high background noise, due to the large amount of...AT-rich DNA and the length of the amplicon and it has a higher rate of false positives (Colborn, 2008; ... used to accurately call copy numbers, though it has been shown to identify trends correctly (Muhamad,...

... disrupting normal brain function. Chemogenetics has become a popular tool in the field as it provides...chemogenetics are instead that it is noninvasive and has longer-lasting effects. Building your chemogenetic... agonist for muscarinic receptor derived DREADDs has been clozapine-N-oxide (CNO). However, concerns have...are already approved for clinical use. Varenicline has been further modified to create ultrapotent PSEMS...

...In the last decade, the use of degron tags has become increasingly popular for the modulation of endogenously...protein levels can return to normal once the drug has been removed. The degradation and reversal are rapid... its first use as a protein regulation tool, AID has gone through an additional round of evolution (AID...the system, but the general concept of the system has remained the same. An obvious drawback of the system...

...FIQ or mStayGold (Ando et al., 2023). This variant has similar photostability and brightness compared to... of AAV9-X1.1 to that of AAV1. This viral vector has brain endothelial cell-specific tropism, with slightly...purify or image a recombinant protein, the FLAG tag has proven itself a good option for essentially all applications...western blots, though the original parent clone (M2) has been widely used in a variety of applications. Find...

...community has access to all of the latest resources? In response to this challenge, Addgene has launched... why recent work in the field of cancer research has focused on picking apart the complex cellular pathways...at Vanderbilt University in Nashville, Tennessee has already demonstrated that a number of novel small...

...host cells immediately prior to their transduction has been shown to increase AAV transduction efficiency... such cationic reagent that is commonly used and has been shown to increase transduction efficiency by...single-use aliquots. Never use a polybrene solution that has been thawed more than once. Fibronectin is another...polybrene (e.g., hematopoietic or primary cells), and has been shown to increase transduction efficiency by...

...regulatory elements. To solve this problem, the Zhang lab has previously used SaCas9 (1053 aa/3.2 kb) with AAV ... these options (2.7 kb with 24 nt guides), so it has the potential to eclipse any of the CRISPR nucleases...THE ORIGINAL NgAgo ARTICLE DISCUSSED IN THIS POST HAS BEEN RETRACTED AND FOLLOW UP STUDIES HAVE FAILED ...be the best option for HDR, but this possibility has not been experimentally confirmed. Researchers have...

...for the presence of an insert. Traditionally this has been done with restriction enzyme digest; however... Find Empty Backbones at Addgene Each approach has advantages and drawbacks, which are summarized in...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...

...Fluorescence is the emission of light by a substance that has absorbed light. The emitted light is at a longer ...protein Excitation & Emission (ex/em): Each FP has its unique ex/em peak. Therefore, choose FPs that...detect the emission. For example, if your microscope has only two lasers, at 488nm and 561nm, you will not...Some proteins are very dim (e.g. TagRFP657, which has a brightness of 0.1) and this should be taken into...