...the Mobile CRISPRi cassette flanked by left and right Tn7 end sequences. Donor bacteria with a chromosomal...scheme. Mobile-CRISPRi integrates downstream of the highly conserved glutamine-fructose-6-phosphate aminotransferase...cell, the CRISPRi DNA (purple) flanked by left and right Tn7 end sequences (green) integrates downstream ...mirabilis, had only ~1 transfer per 1,000,000 colonies. Higher transfer rates are better suited for genome-wide...

...of them: Characteristic ESC ASC Self-renewal High Low once isolated from the human body Usage Universal...specific cell types can be produced Ethical concern High. Embryos are destroyed during ESC isolation. Low...post-transplantation rejection when used therapeutically High Low (if ASCs are isolated from the patient) For...each other in many aspects. Like ESCs, iPSCs have a high self-renewal rate and can be used universally to...

...At Addgene, we've set out to fill this gap by highlighting that researchers learn many transferable skills...the lab and by offering advice on areas where you might need some help. Today in our transferable skills...Although this approach to negotiation is the most straightforward, it can mean that everyone leaves disappointed...

...but, to start, let's review why NgAgo is in the spotlight and take a moment to remember that NgAgo has only...a single experiment. Also, the NGG PAM sequence might not always be available at an optimal distance from...mean the technology is dead in the water. On the bright side, successful indel generation and epitope knockin...supposedly alien 5’-phosphorylated ssDNA guides, which might be rapidly degraded by the cellular machinery, leaving...

...Cas effectors to light. One lesser-known Cas is waiting for its chance in the spotlight — Cas3. Cas3 can...Cas effectors can’t, while maintaining relatively high efficiency. These alterations are achieved through.... E., Bravo, J. P., Ramos, D., Hibshman, G. N., Wright, J. T., & Taylor, D. W. (2023). Structural snapshots...

...added to media, buffer, etc., which will bind and light up a tagged protein for imaging. Given its popularity... like the SMASh tag does. However, it is a straightforward system – HaloTag and its PROTAC are all that...tag Are all tags created equal? No! Choosing the ‘right’ tag does depend a lot on your unique experimental...function is easily perturbed by tags, then a small tag might be the way to go (dTAG/SMASh). If you don’t want...

...being used to perform genome-wide screens and highlight some of the reagents that have been made publicly... library. Choosing the CRISPR library that is right for you There are several factors to consider when... Findings from this type of experiment can shed light on the mechanism by which cells gain resistance ...

...are studying a novel gene, is not always a straightforward process. Rather than try to directly detect...cells that are expressing your gene of interest at high levels. Unlike vectors that express screenable or.... (J Virol. 1988 Aug;62(8):2636-43.) PubMed. 3. Highly Efficient Multicistronic Lentiviral Vectorswith...):845-60. doi: 10.1089/hum.2008.188.) PubMed. 4. High cleavage efficiency of a 2A peptide derived from...

..., so it is pretty important to get this process right the first time. I’ll lay out some guidelines here...transformation process The process itself is straightforward. When your agrobacterial cultures are ready...s flowers inside. 50 mL falcon tubes or plastic weigh boats are both great options for this, depending... et al. (2007) ‘Improved Gateway Binary Vectors: High-Performance Vectors for Creation of Fusion Constructs...

...contributed by guest blogger Tessa Cressey. The highly pathogenic Ebola virus belongs to the group of ...necessity for working with this virus under the highest biosafety level conditions, BSL-4. In this regard..., it is important to choose cell lines that are highly transfectable.  The amounts of each plasmid transfected...vary between labs, initial titration experiments might be helpful to achieve optimal results. It is important...

...the unknown sample’s measurement is too high, start with a higher concentration of standard or make smaller...range is the linear span between the lowest and highest concentrations that the curve can accurately measure...labmates to get a sense of the concentrations you might expect to see for the unknowns in your experiment...

...cell cycle rates; rapidly diving cells will have higher HR frequencies than quiescent cells. It is important...ends of the ssDNA to find a repair template. The highest HDR efficiency is observed when inserts are within... need to incorporate homologous sequence to the right and/or left of the knock-in site. The length of ...: 387-390 (2020). 10.1016/j.jbiotec.2019.11.010 Wright, D. W., Shah, S. S., Heyer, W. D. Homologous recombination...

...samples that come through our doors. It would be a nightmare if we didn’t have a simple way to track all of....2%) transform the first time and can be stored right away. Every week, the lab performs an average of...bacterial glycerol stocks on Fridays. From each overnight culture, we prepare three sets of 2D barcoded ...for a way to organize samples within your lab, we highly recommend using barcodes! Barcoded tubes are easily...

...neuronal networks Double inverted vectors expressing light inducible ion channels (plasmid # 20298 for example...manipulations in vivo, FLEx vectors expressing the light activatable ion channel under the control of Cre...proteins (e.g. postsynaptic PSD95-mcherry) are used in high concentrations in combination with low concentrations...vectors expressing the required recombinase. The high concentration of FLEx vectors guarantees that most...

...that each paper taught me a few things and also highlighted a few dozen things that I didn’t know about. ... whatever time you have. If you’re like me, you might be surprised at how much you can get done between...article. This is normal and it means you’re on the right track!  Get feedback After you’re done editing, ...

...While there is a dose-response relationship (the higher the exposure, the greater the risk of disease) ...developments recently is a new blood test for HMGB1, the high mobility group box 1 protein. Normally localized... test to see how well treatments are working to fight the cancer.  Beyond early modifications to standard...mechanisms) that work through body’s immune system to fight cancer. However, there are many different forms ...

...where the brain fails to divide properly into the right and left hemisphere. Addgene has several plasmids...Spaghetti squash (Sqh) is the Drosophila Regulatory Light Chain (RMLC) of nonmuscle myosin II (Karess et al...PMC2395242. Jordan, Pascale, and Roger Karess. "Myosin light chain–activating phosphorylation sites are required...12490562. Karess, Roger E., et al. "The regulatory light chain of nonmuscle myosin is encoded by spaghetti-squash...

...times during a 10 minute conversation. Leaving a tight-knit team like that can and will be a little gut-wrenching...camaraderie again in a non-academic career. You might not be in a lab anymore, but you will very likely...academia provide a formal training regime and the right set of tools to help you do your job as well as ...lab whether you realize it or not. These skills might include collaborating, managing, and communicating...

...team originally suspected that this new Cas7-11 might be phylogenetically classified as the more complex...Site-specific Targeting Elements (PASTE). Finding the right integrase for this new approach proved a challenge...is more specific and efficient than HITI and has higher purity of insertion than both HITI and HDR. PASTE...indels and no off-target effects, giving PASTE a high-fidelity rate. Both Cas7-11 and PASTE open up multitudes...

... intensity was classified into four groups (4; brighter than control, 3; same as control (Cetn1/sgRNA1...sgRNA1), 2; darker than control, 1; very dark). Brighter fluorescence indicates a more successful gRNA design...your cells under a fluorescence microscope. The higher the amount of fluorescence, the better that gRNA...been inserted or deleted. Each lab will use a slightly different method to genotype their pups. Taking...

... viruses to study. Interestingly, due to certain high profile problems surrounding cell culture over the...but these days it’s never been easier to conduct high quality work in cell culture. The problems with ... Fortunately it has never been easier to conduct high quality cell culture. Biorepositories around the...AstraZeneca-MedImmune, and Infors & Applikon. Due to his high level of experience he now travels around Europe...

...optimized for each specific virus in order to generate high quality preparations. After production, all virus...obtained using ddPCR are generally two to three fold higher than those achieved using the standard qPCR method...performed by ddPCR.  Quality Control Addgene ensures high quality viral vectors by optimizing and standardizing...staining or SYPRO Ruby staining and the molecular weight and relative intensity of the viral capsid proteins...

...choosing a quality mentor, and rightfully so! They can provide you with high-level career advice, open doors...won’t be very proficient afterwards. Below we highlight how to prepare and things to keep an eye out for...

...news briefing, to quick notes, to finances, to flights & ordering food - everything is covered.   So why...for a large institution or company, site licenses might be available as well. In most cases this applies...are collaborating with other labs or clients, you might need share data with them including reports and ...does the ELN provider protect and respect your IP rights and data ownership? IP and data ownership should...

... issue of “tiny vector, big transgene” by just straight up packaging an oversized genome of ~9 kb into...Generally an overlapping split vector has equal or higher transgene expression when compared to using a single...of non-endogenous sequences, such as a 77bp long highly recombinogenic homology sequence derived from the...method While the hybrid dual vector system has a slight advantage over the overlapping and trans-splicing...

...overview of plant genome engineering using CRISPR, highlight the specific modifications to CRISPR machinery...Lab.  As with all plasmids in the repository, we highly recommend reading the associated publications or...10.1104/pp.15.00636 Ma X, Zhang X, Liu H, Li Z (2020) Highly efficient DNA-free plant genome editing using virally...

...developed, which differ in the number, stability, and brightness of the fluorescent proteins used in the system...Brainbow 3.0 (Cai et al., 2013), incorporates the highly photostable fluorophores mOrange2, EGFP, and mKate2...system to boost XFP expression. The increased brightness of Tetbow allows this system to be used to trace...Sakaguchi, Richi, Marcus N. Leiwe, and Takeshi Imai. "Bright multicolor labeling of neuronal circuits with fluorescent...

...Biotin protein ligase (BPL) is responsible for this highly specific post-translational protein modification...photoreactive arylazide group. Under strong visible light, the arylazide group becomes reactive and forms ...: 20353954. 5. Kay, B., Thai, S., & Volgina, V. “High-Throughput Biotinylation of Proteins.” Methods Mol...PMID: 2388593 7. Holden, M. A., & Cremer, P. S. “Light activated patterning of dye-labeled molecules on...

...to make them serve you well. The happy byproduct might just be a career opportunity. The focus of this...content. Create a Google.slides or Powerpoint file right now to capture interesting key results, striking...need to define a term, but are worried that this might sound condescending, consider explaining the term...Additional Resources Joanne's "How to Choose the Right Next Lab or Workplace" On YouTube Joanne's 10 Things...

... can find his original blog post below! --Ed.  Eight years ago, I decided to write a "how to" manual ... and mentoring to several people that found my insights helpful. All went on to get faculty positions ...potential colleagues study. Think about how you might collaborate or, at the absolute minimum, can appreciate...free!) contains strategies, hints, anecdotes, and insights. After I wrote the first version of my manual,...