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...host's replication machinery for elongation and termination. These types of plasmids tend to be high copy...synthesizes a new DNA strand at each replication fork. Termination sequences present in the chromosomal DNA ensure... although ATP does get hydrolyzed over time, inactivating the protein binding it (Grimwade et al., 2018...replication and balance population growth against sustainability. Plasmid DNA replication and regulation Plasmids...which replication of the plasmid proceeds (and terminates) similarly to the bacterial chromosome. Since.... Thanks, stringent regulation! This post was originally written by Jason Niehaus in December 2015 and...Leonard, A. C. (2018). Origin recognition is the predominant role for DnaA-ATP in initiation of chromosome...

...nature can inform us on how to create better recombinant vectors. In this blog post, we’ll define plasmid...harness those features to create broad host range recombinant vectors.  A plasmid’s host range is determined...phylogenetic groups (Jain and Srivastava 2013).  Recombinant vectors that are made from broad host range plasmids...host-independent   Building broad host range recombinant vectors Replicons, which consist of a plasmid...broad host range plasmids can be used to create recombinant vectors that can replicate in different bacteria...to incorporating broad host range replicons, recombinant vectors can be modified further to increase the...cloning sites (Keen 1988).  Find broad host range recombinant vectors at Addgene!   References and resources...

...guests, Melina Fan, Kenneth Fan, and Benjie Chen! Melina Fan is a co-founder of Addgene. Melina received... podcast, we sit down with Addgene’s Founders - Melina Fan, Ken Fan, and Benjie Chen. We learn what it...University, where she studied diabetes and metabolism. Melina has worked at Genetics Institute in Cambridge, ... led business efforts at Addgene as the Chief Financial Officer and Chief Operating Officer. Prior to ...

... requires an N-terminal consensus sequence (MG-) and prenylation requires a C-terminal consensus sequence... filter sets on a fluorescence microscope, and finally (iv) good toleration in fusion proteins. Since ... introducing two mutations (F64L, S65T) in the original jellyfish GFP (AvGFP) to create enhanced GFP (... loop-structure (Querido and Chartrand, 2008). Finally, a nuclease-deficient Cas9 can bind loci in the...GFP is fused to these sequences. When N- and C-terminal fusions are not tolerated the alternative is to... subunit fusions that do not tolerate N- and C-terminal fusions (Adjobo-Hermans, 2011). Structural information...

...markers and homologous recombination As an alternative, endogenous homologous recombination enzymes can also...enzymes and those that use endogenous homologous recombination. Modifying bacterial genomes using exogenous...exchange requires two sequential homologous recombination events, but because these events are rare, each... ability to grow on sucrose, perhaps by sacB inactivation.  Bacterial toxins for counter-selection In ...mix. Introduction of the strong transcriptional terminator BBa_B1002 (from IGEM) upstream of the MCS to ...genetic engineering in bacteria using homologous recombination." Current protocols in molecular biology 106.1...

...focus on defining these resources and not on the financial aspects. Money often carries an emotional weight...administrative services, managers, and techs’ financial books.  To find personnel salary ranges, go to...using animals, then that’s a cost that you can eliminate from your budget. Every cut you make signifies...signifies that you've made a conscious effort to eliminate some of the excessive costs in your lab. That in itself...specific aims. 2. Then make decisions based on financial values. All too often, we see early investigators...save you money in the short term. However, the financial risks associated with old equipment, like service.... Always focus on the science When negotiating finances, instead of solely discussing numerical values...

...single-stranded to double-stranded, and finally, drop once more during a final temperature increase. If there ...are cases in which it is an appropriate choice (Panina, 2018). Look for studies specifically aimed at ...real-time RT-PCR. J. Immunol. Methods 283:291–306.Panina, Y., Germond, A., Masui, S. et al. Validation of...M; Schwarz G; Pfaffl MW. 2003. Standardized determination of real-time PCR efficiency from a single reaction...Nucleic Acids Res. 31:e122.Thelwell N; Millington S; Solinas A; Booth J; Brown T. Mode of action and application..., Dong X, Zhang M, Hu G, Fang F. Fudenine, a C-terminal truncated rat homologue of mouse prominin, is ...11237753. Primer design resources PrimerBlastDINAmelMfoldBLASTEnsembl More resources on the Addgene blog...

...Executive Director, Joanne Kamens, and founders, Melina Fan, Ken Fan, and Benjie Chen. Last week, the Addgene...weren’t able to join us in person. Joanne Kamens, Melina Fan, and Ken Fan each spoke about what Addgene ...mentioned Addgene’s incredible growth and our determination to keep materials as affordable and accessible...without whom Addgene could not do what it does.  Melina talked about her grandfather’s humble beginnings... heartfelt portion of the entire night was when Melina and Ken called their parents up to the podium to... great (maybe too great!) time. Ten years ago, Melina Fan had an idea and her family helped bring it ...

...these devastating afflictions. Elia Di Schiavi - Spinal muscular atrophy Dr. Elia Di Schiavi is a Research...Bioresources in Naples, Italy. His group works on spinal muscular atrophy, a disease in which disruption...of the smn1 gene results in the degeneration of spinal cord motor neurons. This disease is estimated to...While disruption of the smn1 gene is known to cause spinal muscular atrophy, the molecular mechanisms behind...clinic and thereby restore motor neuron function in spinal muscular atrophy patients. Noushin Nabavi - Peritoneal...Systematic review of incidence and prevalence of spinal muscular atrophy (SMA)." European Journal of Paediatric...

...genetic tools: activators, inhibitors, sensors, recombinases, and more. With the help of adeno-associated...continuously refined and developed so there are many combinations to choose from for your experiments! Late last... aimed at brainstorming strategies for using combinations of AAV-based molecular tools for targeted neuronal...specific expression of molecular tools and sensors Combinations of classical genetic tools, for example Cre/...restricted light exposure to a specific area in combination with delivery of AAV at the injection site results...specificity of the targeted cell population.  Combinatorial strategies can also be used for retrograde tracing...

...Like everyone else in the world, I (Wenning) was fascinated by CRISPR and rushed to test it. I still remember...the day, March 16, 2014, that I got my first preliminary results from a CRISPR genome editing experiment...was shared by many others around the world. Yes, finally, we have a technology, CRISPR, that is simple in...conventional gene targeting, the cost may be $50,000. Finally, compared to conventional methods of genome engineering...advice from John Doench’s gRNA design blog post. Finally, always remember that you are working with RNAs... active sgRNAs for CRISPR-Cas9–mediated gene inactivation. Nat Biotechnol 32:1262–1267 . https://doi.org...in vivo genome editing in mouse embryos using recombinant adeno-associated viruses. Nat Commun 9: . https...

...you are studying the effect of a new tau protein kinase inhibitor on slowing diseases progression in an...group of mice, the experimental group, with the new kinase inhibitor and a second group, the positive control...control group, with an alternative but well-studied kinase known to prevent tau hyperphosphorylation. You ...experimental protocol but does not receive either protein kinase inhibitor. Since the negative control group does...variability. If one were studying the effects of tau kinase inhibitors on mice, for example, it would be better...protocol and try to find a way to reduce, if not eliminate, the noise. This can be done either by modifying...labeling occurs (Burry 2011). Complex experiments Finally, additional controls may be required for more complex...

...Addgene's own empty lentiviral backbones New recombinant antibodies for Glypicans DNA editing with STITCHR...tagging: Choose untagged or add a versatile N- or C-terminal HA tag. Selectable and stable: A puromycin resistance...11012), and counterstained with DAPI. New recombinant antibodies targeting Glypicans By Ashley Waldron...) recently released a set of highly-specific recombinant antibodies against three of the six human glypican... et al., 2024). While dCas9 has been used in combination with proximity labeling enzymes before, such ..., Bosch, J. A., Sanchez, A. D., Udeshi, N. D., Svinkina, T., Carr, S. A., Feldman, J. L., Perrimon, N....parvalbumin (PV), somatostatin (SST), vasoactive intestinal peptide (VIP), or lysosomal-associated membrane...

...expression.  Finally, the risks of potential toxicity and/or off-target effects (e.g. recombinase-dependent...rather than relying on other systems as proxies.  Finally, the size and receptor interactions of a serotype...Nonhuman Primates after Intramuscular Injection of Recombinant Adeno-Associated Virus. Journal of Virology, ...promoter results in robust transduction in the rat spinal cord following injury. Gene Therapy, 26(5), Article...adeno-associated viral vector-mediated transduction of the corticospinal tract: Comparison of four promoters. Gene Therapy...doi.org/10.5607/en20006 Rolland, A.-S., Kareva, T., Yarygina, O., Kholodilov, N., & Burke, R. E. (2016). Expression...

...multicolor labeling is a powerful solution for discriminating between neurons for light microscopy-based ... introduced, stochasticity will result in a combinatorial expression of these three genes with different...inherently low. This is because the stochastic and combinatorial expression of fluorescent proteins is only possible...overcome the limited expression levels of XFPs in the original method, we used the Tetracycline trans-activator... long, as too much XFP is toxic to the cells. Inadequate expression levels of XFPs - Reduce the amount...Livet, Jean, et al. "Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous...

...pENN.AAV.hSyn.HI.eGFP-Cre.WPRE.SV40 AAV2 Recombinase James M. Wilson New serotype pENN.AAV.hSyn.Cre.WPRE.hGH AAV2 Recombinase James M...AAVrg Recombinase James M. Wilson New serotype pENN.AAV.CamKII 0.4.Cre.SV40 AAVrg Recombinase James ...Ofer Yizhar New serotype pAAV-syn-Flpo AAVrg Recombinase Ian Wickersham New viral prep pENN.AAV.hSyn.HI.eGFP-Cre.WPRE.SV40...-iCre(R297T)-BGHpA (Alias: CN4825) AAV-PHPeB Recombinase Jonathan Ting New viral prep AiP15140: pAAV-AiE0873m...

...protocols, including vector-insert joining for recombinant plasmid construction (restriction cloning), adaptor...can reliably detect single-base differences. The original creators of this technique visualized the ligation... have been generally found to have a higher discrimination at the upstream side of the ligation junction...including Taq DNA ligase, are naturally able to discriminate against ligating substrates containing base ... at elevated temperatures, allowing further discrimination by incubating the ligation at a temperature... fidelity ligation can be achieved through a combination of the intrinsic selectivity of the ligase active...method allowed screening of all possible base combinations (Watson-Crick and mismatched) around the ligation...

...CRISPR/Cas9 gene edit. I wanted to inactivate the gene BRAF (a kinase implicated in several human cancers... really need to use FACS (like they did in the original publication). I thought I could see GFP expression...against GFP expression, but I didn’t. 2. In the original publication they see GFP expression in about 20%... cells at an MOI of 0.3, as recommended by the original publication. Pro-Tip: 1. Use multiple gRNAs to...human line, using methods based on homologous recombination (Rago et al., 2007). Using CRISPR, I was able... active sgRNAs for CRISPR-Cas9-mediated gene inactivation. Doench JG, Hartenian E, Graham DB, Tothova ...

... are two types of Class 1 TEs: those with long terminal repeats (LTRs) and those without (non-LTR TEs)...contain two open reading frames (ORFs) which often terminate with a poly(A). ORF2 encodes endonuclease and ...transposase enzyme binds the transposon’s long terminal repeats (LTRs), induces a double-stranded break...synthetic transposable element developed from inactivated Tc1/mariner transposons found in fish (Sandoval-Villegas...it leaves behind the CAG DNA footprint from its terminal sequences at the excision site after cleavage ...medaka fish because its insertion into the fish’s tyrosinase gene caused albinism (Koga et al., 1996). Unlike...Tol2 or miniTol2 is a truncated version of the original Tol2 and has ~3-fold increase in transposition...

...inventory of recombinases and recombinase-dependent vectors. We recently released several recombinases in various...Calcium sensors and GABA sensors Optogenetics Recombinases And more! Control AAV For many AAV experiments...densely-labeled neurons because background fluorescence from inactive neurons is reduced. This variant has an approximately...pAAV-CamKIIa-ChrimsonR-mScarlet-KV2.1 (124651-AAV9) Recombinase AAV Targeting and manipulating specific, genetically-defined...