...am and Joanne didn’t have to start answering until 5 pm). The ability to see the questions first makes ...Joanne enough for all the work she put into this. 5. Reddit forums are evergreen! With all the great questions...not too worried about selling yourself and really do just want to get information about yourself and your...where users can participate in forum discussions about a wide variety of topics. Individual forums are ...where users can ask designated scientists anything about a particular topic. Given Joanne’s expertise in... is well moderated. One of our original worries about sharing science advice on Reddit was that we’d be...answering questions. r/Science has pretty strict rules about not being too sales-y in posts which falls in line...

...the forms tRNA-gRNA and tRNA-gRNA-tRNA, with short 5’ spacer sequences preceding gRNAs, to test for gRNA... predicted with no addition of nucleotides to the 5’ spacers. The 3’ ends were modified slightly with ... in the tRNA-gRNA-tRNA constructs. gRNAs with any 5’ leading nucleotide could be processed, a distinct...other methods that favor certain nucleotides at the 5’ end. PTG construct schematics. Scissors indicate...Proc Natl Acad Sci U S A. 2015 Mar 17;112(11):3570-5. doi: 10.1073/pnas.1420294112. Epub 2015 Mar 2. PubMed...with limited packaging capacity. Still confused about the various tRNA expression strategies? No worries... Addgene. If you have any questions or thoughts about this technology, leave them in the comments below...

... audience questions and an audience member asks a 5 minute question that is really a speech? You have ...event is what is important. Choose 4 panelists (maybe 5) and no more. Too many panelists will negatively affect...going to be good at talking, so they will easily take 5 minutes to introduce themselves and, voila, 1/2 of... are nice if you can afford them). I am talking about making sure the audience is interested, educated...of the stories or topics you know they can speak about. There should be some spontaneity, but having a ...location and parking information and remind them about the event as it approaches. Room set-up and logistics... make or break the discussion section. And what about those audience members that want to take over? It...

...restriction sites of the MCS are only regenerated at the 5’ and 3’ termini of the ligation product, and the restriction...between them, but rather, can only be added to the 5’ or 3’ terminus using the newly generated MCS. MXS-chaining... control whether the third module is added to the 5’ terminus or the 3’ terminus of the ligation product...several hundred kilobases. Nature methods. 2009. May;6(5):343–345. Pubmed PMID: 19363495. 4. Josse J, Kaiser.... 1961. March;236:864–875. Pubmed PMID: 13790780. 5. Shetty RP, Endy D, Knight TF. Engineering BioBrick... parts. Journal of biological engineering. 2008;2:5. Pubmed PMID: 18410688. PubMed Central PMCID: PMC2373286...Resources on the Addgene Blog Read about Gibson Assembly Learn About Other Plasmid Cloning Techniques Plasmids...

...without worry, as I had never worked the traditional “9-5” job. Prior to this, the only work experience I had...Science Communication function at Addgene. Working a 9-5 As it turned out, Addgene uses something called “core...accustomed to the routine, I realized that the “9-5” was not as scary as it seemed. The transition into...intimidating to me, as I had a hard time thinking about how I could fill this time. I was relieved to find...attend to other commitments, without feeling guilty about missing work. As long as my work was finished on...

...It contains short promoter sequences at its 3’ and 5’ ends that are crucial cis-acting signals for genome...version of the viral genome. It contains the 3’ and 5’ ends of the genome which are required for replication...transcription by the EBOV polymerase (reviewed in 2, 5). The minigenome is capable of being replicated and...transcription: NP, VP35, VP30, and L (reviewed in 5). The RNA genome is encapsidated by the nucleoprotein...or using cells that stably express T7 (e.g. BSR T7/5 cells) (1). We hope that minigenomes will greatly ...PubMed PMID: 9971816. PubMed Central PMCID: PMC104478. 5. Mühlberger, Elke. "Filovirus replication and transcription...Microbiology, National Emerging Infectious Disease Laboratories.  She is interested in genome replication and...

...modular inserts that span the human adenovirus serotype 5 (HAdV5) genome that can be assembled in a one-tube...information about: Adenoviral components Adenoviral plasmid construction Frequently asked questions about adenovirus...

...spinal cord following injury. Gene Therapy, 26(5), Article 5. https://doi.org/10.1038/s41434-019-0075-6 Issa... Figure 2: Comparison of AAV serotypes 1, 2, 5, 6, 8, and 9 in two different brain regions.  Image...Applications in Gene Therapy: An Overview. Cells, 12(5), 1–41. https://doi.org/10.3390/cells12050785 Murlidharan...Tropism and Axonal Transport of AAV Serotypes 1, 2, 5, 6, 8 and 9 in the Mouse Brain. PLOS ONE, 8(9), e76310...Applications in Gene Therapy: An Overview. Cells, 12(5), 1–41. https://doi.org/10.3390/cells12050785 Kanaan...Perspectives of AAV Tropism Modification. Pharmaceutics, 13(5), 750. https://doi.org/10.3390/pharmaceutics13050750... of adeno-associated viral vector serotypes 1, 2, 5, 8 and 9 in marmoset, mouse and macaque cerebral cortex...

...researchers. Assuming 5 hours of hands-on time to make each plasmid, we would have saved 5 million hours through...sharing. How much is 5 million hours? A colossal 547 years! While we are very excited about reaching this milestone...to picture (see the picture of our shipping room above), but as Joanne Kamens, Addgene’s executive director...

...retroviral transfer plasmids that have inserts. ID Plasmid 5'LTR Description PI 1780 pBABE-neo largeTcDNA MoMLV... information about: γ-Retroviral packaging systems Frequently asked questions (FAQ) about retrovirus Popular...Learn about retroviral plasmids at Addgene, including popular packaging, envelope, and transfer plasmids...

...your results. (Figure 5). The digestion product (Figure 4, lanes 0.3 % 3.0, Figure 5, lane 2) is faint, ...incredulously, “You seriously didn’t add 3 microliters of 5 mM star anise?” This is something I was expecting ...and from individual cell expansions (monoclonal, 1-5). Wild-type A549 cells do not express CAs9 and are...cells transduced by the control GFP-gRNA (Figure 4, 5). When the gel was overexposed, I could even also ... the undigested control (Figure 4, lane 0, Figure 5, lane 1) is what confirms it’s a real digestion product...product and not another PCR product. Figure 5: gRNA-targeted BRAF sites were edited in A549 cells...the editing efficiency, and it did not (Figure 4). 5. In theory you could use densitometric analysis to...

...mediated saturation genome editing of human TP53 exon 5–8. Phagemid Synuclein VHH Immune Library Screening...plasmids that are available to industry. Learn more about the available plasmids and how to order them. Distribution...Information page for more information. To learn more about Addgene’s MTA process please visit our Help Center...

...inexpensive and can be 5’-phosphorylated in house using a simple PNK reaction or can be purchased 5’-modified from...Therefore, researchers must co-transfect cells with 5’-P-ssDNA guides and an NgAgo expression plasmid to... could be the instability of the supposedly alien 5’-phosphorylated ssDNA guides, which might be rapidly...endonuclease) protein that can form a complex with a 5’-P-ssDNA guide at room temperature. Until that time...technology. To dig more into what researchers felt about NgAgo, I conducted a survey and asked researchers...successful, and the second section asked basic questions about the NgAgo protocols used by respondents (transfection...27136078. 2. Burgess, Shawn., et al. "Questions about NgAgo." Proteins & Cell (2016). 3. "NgAgo: Genome-editing...

...disease, as well as those that play into late onset AD (5).     CRISPR/Cas9 corrects Alzheimer’s disease...Front. Oncol. https://doi.org/10.3389/fonc.2020.01387 5. Rohn et al. 2018. “The Potential of CRISPR/Cas9 Gene...Use of CRISPR/Cas9." Cell Stem Cell. 13, 659-662 (5 December 2013). 8. Gerald Schwank et al. "Functional... Fibrosis Patients." Cell Stem Cell. 13, 653-658 (5 December 2013).    ...therapeutic.” There are many fundamental questions about CRISPR/Cas biology still left to answer as well,...

...crRNA DNA 5' NGG Specific blunt ended DSB in target DNA Cpf1 RuvC-like crRNA DNA 5' TTN Specific...Specific DSB in target DNA with 5' overhangs Cas13a (C2c2) 2x HEPN crRNA RNA 3' A, U, or C (not required...LwaCas13a is also amenable to multiplexing; expressing 5 gRNAs simultaneously produces comparable gene knockdown...28475872.  Find plasmids from this paper at Addgene.  5. Abudayyeh, Omar O., et al. “RNA targeting with CRISPR-Cas13...potential applications of Cas13a given what we know about its structure and function? For starters, Cas13a... Check Out Our CRISPR Featured Topic Page Learn about Anti-CRISPRs Multiplex Genome Editing with Cpf1 ...

...modifications, which can all potentially affect function [5]. We and others have reported that FP cysteine residues...properly exiting the ER and fluorescing (see Figure 2) [5].  When using FPs containing two cysteine residues...FP-fusion proteins. Our studies and those of others (5, 6, 7, 10, 11) highlight the very real consequences...published oxidative optimized palette of FPs (moxFPs) [5]. The moxFPs overcome the inherent problems of popular...PMID: 23971632. Pubmed Central PMCID: PMC3806368. 5. Costantini, Lindsey M., et al. "A palette of fluorescent...moxFPs over standard FPs for the reasons listed above. In addition, because the moxFPs are highly monomeric...Medicine. Her doctoral research was conducted in the laboratory of Erik Lee Snapp and focused on optimizing fluorescent...

...Brandeis University. 5 easy steps to forming your own peer mentoring group Find 5-8 colleagues that you...these programs typically consist of 1-2 mentors and 3-5 mentees who meet together for an 8 month formal program...available to go around. My advice has been to find 5 interested peers and form your own peer mentoring ...for Scientists Series. I have been thinking a lot about Mentoring for over 10 years. Many successful scientists...Medical School researcher Connie Cepko to learn about her mentoring style. While working on this project...postdocs I have started to talk to trainees more about peer mentoring. They want to find mentors to help... questions I get is, “What are we going to talk about?” There are an infinite number of ideas on the internet...

...Methods, 20(6), 908–917. doi: 10.1038/s41592-023-01880-5. PMID: 37188954. Preprint available on bioRxiv – doi...activation induces cFos::GFP-iPAK4 at 3 or 6 h. Scale bar 5 µm. Images adapted from Lin et al., 2023 bioRxiv, ...intracellular recordings. Nature Biotechnology, 41(5), 631–639. doi: 10.1038/s41587-022-01524-7. PMID: ... slice (70 μm) immunohistochemistry (IHC) (Figure 5). PSD-95 is a scaffolding protein known for its roles... on their webpage: mAb3D Atlas.    Figure 5: Delipidated mouse brain tissue was stained with Anti-PSD...section of the Addgene antibody page, and learn more about the whole-brain reference atlas efforts, including...

...type targets. Comparatively, detection limits of about 5% mutant targets have been reported for mismatch...resulting from non-homologous end joining (NHEJ) (2-5), depending on your desired genome edit. Since exploitation...PMID: 25964300. PubMed Central PMCID: PMC4477672. 5. Sedlak, R. H. et al. Digital detection of endonuclease... triggered by engineered nucleases. G3 (Bethesda) 5, 407–415 (2015). PubMed PMID: 25566793. PubMed Central...advantages over mismatch nuclease assays that will be elaborated below. Digital PCR Digital PCR (dPCR) involves...

... kinase C Protor Also known as PRR5; proline rich 5 PTEN Phosphatase and tensin homolog Rictor RPTOR independent... the cell and regulates the switch from anabolic to catabolic processes. Active mTORC1 promotes protein...mTOR pathway. enhanced signaling of mTOR, a key metabolic regulator, correlates with poor prognosis in many...ammalian t arget o f r apamycin (mTOR) is a key metabolic regulator controlling cell growth and proliferation...energy-intensive processes. mTORC1 also blocks catabolic processes including autophagy and lysosome biogenesis...