...modifications and homologous sequences are encoded at the 5’ end. First design the template binding region, with...insertion site. Encode half the new sequence at the 5’ end of each primer, with an overlap for homologous...primer by adding a short sequence complementary to the 5’ end of the reverse primer. Mutagenesis - Mutagenesis...codon and homologous region can be included at the 5’ end of either primer. Subcloning - To move a gene...recombination events to assemble the final plasmid. At least 5 fragments can be correctly assembled using IVA cloning...our Plasmids 101 blog posts Read our blog post on 5 factors to consider before choosing a cloning method... Watson and Javier García-Nafría from the MRC Laboratory of Molecular Biology. Plasmid cloning is an essential...

... Click on the player below to listen to a quick (~5 min) Hot Plasmids podcast that introduces you to 4...entire Hot Plasmids episode! We generally publish 5-10 Hot Plasmid articles with each of our newsletters...Advice on Picking a Graduate Lab and Project Check Out 5 Science Videos from Tom McFadden Resources at Addgene.org...this new way to learn about plasmid technologies. If you have any comments about the Hot Plasmids Podcast...with these efforts, you’ll find new ways to learn about science careers, lab protocols, and, of course, ...each one, plug in some headphones to learn more about the latest and greatest plasmids through this podcast...

...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...

...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...

...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...

...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...

...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,...

...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...

...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 ...

...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...

...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 ...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...the editing efficiency, and it did not (Figure 4). 5. In theory you could use densitometric analysis to...designed (listed above) worked well and I got clear bands using the conditions above. 2. Design your primers...essential for experiments but that nobody talks about. After months of troubleshooting, those people who...

... Trypanosoma brucei Cross 34564 pMSCVpuro-Cre Cre 5'LTR Retroviral Ge 34565 pMSCVhygro-Cre Cre and HygroR...HygroR coexpression Retroviral Ge 34568 WZLneo-Cre Cre 5'LTR Retroviral Ge 35619 pTAT-CRE Cre fused to HIV-...1027-32. doi: 10.1073/pnas.0610155104. Epub 2007 Jan 5. PubMed . Sauer B, Henderson N. 1988. Site-Specific...Transgenes. Proc Natl Acad Sci. Jul 13;101(28):10380-5. doi:10.1073/pnas.0403954101. Epub 2004 Jul 6. PubMed...Learn about the Cre-Lox system and how it can be used for genetic rearrangements and recombination. Use...

... our 96-format pipeline, we can routinely achieve 5-30% tag knock-in efficiency across different stem ...other suppliers with a typical turnaround time of 5-7 days. The crRNA and tracrRNA are annealed on a PCR...into stem cells. Cells are allowed to recover for 2-5 days and then analyzed for tagging efficiency using... in 96-well format using an Amaxa Shuttle device; 5 days after the transfection, the entire plate was ...commercial suppliers of CRISPR reagents (turnaround time 5-7 days); because the tracrRNA is the same in all csRNP...suspension, transfect and let cells recover for 2-5 days.Note: We use the Nucleofection method for delivery...J Biotechnol 241: 136-146. PubMed PMID: 27845164. 5. Clarke, Ryan, et al. "Enhanced bacterial immunity...

...iPSCs from PBMCs. Most laboratories use Sendai virus to reprogram PBMCs efficiently [5]. However, the Sendai...establishment of virus-free hiPSCs usually require about 5-10 passages. Furthermore, working with Sendai virus...2003. 302(5644): p. 415-9. PubMed PMID: 14564000. 5. Seki, T., S. Yuasa, and K. Fukuda, Generation of ...pluripotent stem cells. Korean J Intern Med, 2014. 29(5): p. 547-57. PubMed PMID: 25228828. 7. Okita, K., ...p53-p21 pathway. Nature, 2009. 460(7259): p. 1132-5. PubMed  PMID: 19668191. PubMed Central PMCID: PMC2917235...source of somatic cells. This translates to only about 16 hiPSC colonies per 100 ml of starting blood sample...

... Hive Beautifully filmed video about colony collapse disorder.     5. Plasmid Rap Plasmid rap video...Science with Tom. In this podcast, we learn more about Tom and pick his brain for advice on how to dive... Rap of Henrietta Lacks Students in Oakland rap about Henrietta Lacks - the African American woman whose...science standards and communicate science. Learn more about Tom on his youtube channel and at sciencewithtom.com...

...proposal successful. Typical proposals consist of 3 to 5 aims, with secondary milestones to meet along the ...statements on the next round of applications. Step 5: Keep writing! Each written grant application builds...first. Additionally, reach out to peers and collaborators who have succeeded at grant writing to see what...