... to limitations in light penetration mentioned above (5). Thick static samples Ex: Fixed tissue sections...providing fast imaging along with optical sectioning (5). One such example is the lattice lightsheet that ... Enzymol 361, 1-33 (2003). PubMed PMID: 12624904. 5. Power RM, Huisken J. A guide to light-sheet fluorescence...call Epi-fluorescence microscopes. (See left image above for an example image). Point Scanning Confocal Microscopy... plane to reach the detector and all light from above or below is excluded. Parallelized Confocal Microscopy...sectioning. Point scanning confocal microscopes (see above) often provide the highest quality images for this...

...contaminant we mixed into the original AAV sample at 5%. Detecting 5% contamination is a good start, but to determine...viral genome sequencing (VGS). Why should you care about viral quality control? AAV vectors are produced ... let us know if you have any specific questions about viral DNA NGS you’d like us to discuss. We are also...or at [email protected] to answer your questions about our quality control process! The images in this...

... the desired gRNA target sequence, as well as the 5’ and 3’ homology arm repair template (Fig. 1, Step...of the same type will produce the same unique SapI 5’ overhang, different combinations of connector and...tagged gene. Most insertions can only be detected by labor-intensive processes like PCR or by evaluating visual...repair after a CRISPR/Cas9 induced break. Learn more about these constructs and the cell lines they’ve been...Additional Resources on the Addgene Blog Learn about Fluorescent Protein Timers Check out Photoactivatable...

...Money with the Baby Gel Box Find 5 Smart Phone Apps for Lab Life Learn About the Evolution of Lab Techniques...Digests Make Your Agarose Gel Other Resources Read about Gel Stains on the BitesizeBio Blog ...

... Neurovirol. 2005;11:82–87. PubMed PMID 15804965. 5. Nassi JJ, Cepko, CL, Born RT, Beier KT. Neuroanatomy... midbrain dopamine neurons. Neuron. 2012 Jun 7;74(5):858-73. Pubmed PMID 22681690.  8, Wickersham IR, ...connected to the neck. Shortly after, we learned about organs and the jobs they perform. This became foundational...part of the answer to many biological questions about the brain. To address this, molecular tools to map...

... in the absence of genome editing (Fig. 2 and 3) (5, 3). Figure 2: Gene activation with nuclease-competent...PMID: 23287722. PubMed Central PMCID: PMC3712628. 5. Dahlman, James E., et al. "Orthogonal gene knockout...of the Addgene Scientific Advisory Board. His laboratory at Columbia University is focused advancing scientific... Listen to Our Interview with Alex Chavez Learn about Sequencing Options for CRISPR Genotyping Cas9 Activators...

... quantitative PCR. Expert Rev Mol Diagn. 2005 Mar;5(2):209-19. doi: 10.1586/14737159.5.2.209. PMID: 15833050...resistance-1 gene (pfmdr1). J Parasitol. 2011 Oct;97(5):939-42. doi: 10.1645/GE-2792.1. Epub 2011 May 9. ...controls must be run in triplicate.  The nice thing about qPCR is that since it measures relative quantification...number, albeit within a range of RQ values as noted above.   In both applications, results can be affected...

...sequence-specific control of gene expression. Cell, 152(5), 1173-1183. (2013). PubMed. Zalatan, J. G. Engineering...successes in metabolic engineering have been limited to the overexpression of various metabolites in Escherichia...species or metabolic pathways. A new study takes us one step closer to the vision of metabolic biofactories...cellular behaviors, such as differentiation or metabolism. Our attempts to manipulate these cellular behaviors...be host-independent, beyond the oft published metabolic engineering realm of S. cerevisiae and E. coli...control via scRNA design is readily apparent in metabolic engineering. The authors elegantly demonstrated...demonstrated the potential of this to control a metabolic network in S. cerevisiae by using three RNA programs ...

...platform, the Jacks lab generated 5 common 30bp overlap sequences (Sites #1-5). Each overlap site encodes a...GMAP-compatible backbones for lentivirus LV 1-5 and retrovirus RV 2-5 were constructed. The authors then used...available at Addgene. Since GMAP assembly utilizes 5 different overlap sites, this technique also allows...different human cell models and identified ~4-5-fold more fitness-related genes than have been found in previous...allowing MAGE in otherwise unmodified bacterial strains (5). MutL is highly conserved in distant relatives of...but be sure to let us know if you'd like to write about your plasmids in a future blog post. No time to ...pooled libraries, but thanks to a deposit from the laboratory of Dr. Ji-Long Liu at Oxford University, we are...

...say? Do you know what’s even better? Going home at 5 o’clock. You’re going to have to talk and interact...interact with...non-scientists! Maybe you’ve just spent 5-10 years learning to “speak science,” and virtually...scientist anymore. Being a scientist is not about what you do--it’s about how you think. I have not worked at ...10 years, but when people ask me about my job, I have no qualms about saying “I’m a scientist.”    What...installment of the Addgene career guide, we’ll think about what causes this culture shock and how to overcome...counterpoints to some of the causes of culture shock above, but after polling some Addgene colleagues, we’ve...This is probably because you don't have to worry about experiments taking longer than expected or dealing...

...expression strategies.” Chem Biol. 2015 May 21;22(5):649-60. PubMed PMID: 26000751. PubMed Central PMCID...Nanotechnology and Therapeutics. Boca Raton: CRC Press, 2014. 5. Hayat, Akhtar and Marty, Jean L. “Aptamer based electrochemical...target metabolite creates an aptamer that will only bind to DFHBI in the presence of that metabolite (Fig...approach less sensitive in most applications. Learn About Fluorescent Biosensors Light-up aptamers (or, fluorescing...fluorophore protein. Years later, Samie Jaffrey’s laboratory took advantage of their deep knowledge of the...Tips for Using FRET in Your Experiments Learn more about GFP Controlling Protein Activity with GFP Resources...

...interactions in living cells. Bioessays. 2012 May; 34(5):341-50. doi: 10.1002/bies.201100098. Sekar RB, Periasamy...protein localizations. J Cell Biol. 2003 Mar 3;160(5):629-33. ...time I heard about FRET during a journal club, my guitarist brain automatically thought about the raised...you would say. The student was of course talking about the now well-known FRET, aka Fluorescence (Förster...other. FRET reduced the scale of our perception about molecular interactions. What is FRET? In their ...Resonance Energy Transfer    Interesting Articles About FRET: Piston DW, Kremers GJ. Fluorescent protein...

...job where people have regular schedules. Work 8 to 5 or 7 to 4 or maybe 7 to 6 and 8 to 10 or whatever ...before you get enmeshed in a serious relationship. #5 Thou shalt not sweat the small stuff The house can...on the Society for Laboratory Automation and Screening (SLAS) Electronic Laboratory Neighborhood.   Work...worry less about how they will handle an emergency and this will make you much more confident about their ...

... efficiencies (Chen et al., 2021). Figure 5: MMR efficiently targets single-base mismatches (left... Complex with Guide RNA and Target DNA. Cell, 156(5), 935–949. https://doi.org/10.1016/j.cell.2014.02.001... primer binding site, starting with a length of about 13 nt (Anzalone et al., 2019). Primer binding sites...of reverse transcriptase template, starting with about 10–16 nucleotides. For longer templates, it is even.... Test multiple nick sites, starting with sites about 50 bp upstream and downstream from the prime editing...

...PubMed PMID: 17690442. 5. Weigel, D. and Glazebrook, J. (2002) Arabidopsis : a laboratory manual. Cold Spring...stock or a colony from an LB plate. In my hands, a 5 mL starter culture inoculated from a colony on a plate...Spring Harbor Laboratory Press. Available at: https://books.google.at/books/about/Arabidopsis.html?id=IfZAMNPWVk4C...

...RNA-guided nucleases.” Nature Biotechnol. 32(11): 1141-5. (2014). PubMed: PMID 252409288. PubMed Central: PMCID... PMID 26060300. PubMed Central: PMCID PMC4466736. 5. Ando, Hiroki, Sebastien Lemire, Diana P. Pires, and...organizations are working to help educate the public about proper antibiotic usage. Although these steps are...laudable, they likely won’t solve the growing problem. Above all, we need new antimicrobials, ideally ones for...used to deliver any of the three systems discussed above, with the caveat that phages are very diverse, and...

... more than two nucleotides in the same position; (5) avoid overhangs with either 0% or 100% GC.) In fact...could be done using overhang sets that broke rules 3—5. Fidelity, it turns out, might depend more on the ...cycling. The Pros and Cons Researchers used the tools above to complete a 52-piece assembly of a 40 kb T7 phage... help from DAD. The 35-piece assembly mentioned above had a remarkably high 71% fidelity, while the 52...

... Therapy Vol. 25 No 5. PubMed PMID: 28391961. PubMed Central PMCID: PMC5417792. 5. Naldini,L., 2015. Gene...famously visited Congress to help increase awareness about the potential of gene therapy!). It would take another...muscular atrophy, neurodegenerative diseases and metabolic diseases are also underway and reported results...

...luciferase protein interact and enable intein splicing (5). Screening with yeast two hybrid systems While initial... Nature. 1989; 340:245-246. PubMed PMID: 2547163. 5. Ozawa, T., Kaihara, A., Sato, M., Tachihara, K., ... Plasmids Read our Blog Post About Yeast Vectors Read our Blog Post About Tagging Yeast Genes ...

...an attL-containing entry vector.   Figure 5: Method C to create an entry clone: Restriction cloning... Hosts. Curr Protoc Protein Sci. 2003 Feb;Chapter 5:Unit 5.17. PubMed PMID:18429245. 3. Ptashne, M. (1992... expression systems. Keep reading to learn more about the Gateway cloning method and its advantages. An... on the Addgene Blog Try Out FastCloning Learn About Methylation and Restriction Enzymes Use Colony PCR...