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  1. Hot Plasmids - December 2019 - CRISPR pooled library, LipoGlo, CRISPRa, and Voltage Imaging Tools

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    Blog Post
    ...plasmid deposit please sign up here.        A retroviral mouse CRISPR knockout library Article contributed...phenotype, and this depends on the method used to introduce the gRNAs into cells. Most CRISPR pooled libraries...issue, Sarah Teichmann and colleagues created a retroviral CRISPR knockout library using gRNAs designed ...designed by the Kosuke Yusa Lab. This retroviral library was used to transduce T helper type 2 (Th2) cells from...differentiation factors as the screen readout. This retroviral mouse CRISPR pooled library can be used for screening...concentration of ApoB-LPs has been shown to have a strong correlation with cardiovascular disease and indirect...
  2. New Neuroscience Tool: The iGluSnFR3 Glutamate Sensor

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    Blog Post
    ... inserted GFP. After characterizing iGluSnFR in vitro, showing that it is selectively activated by glutamate...higher expression levels in bacteria and produces stronger fluorescent signals upon sensing glutamate in ...variants differing in kinetics, affinity, and ∆F/F0. Introduce the two variants in three different membrane-targeting...iGluSnFR3 have higher expression levels and produce stronger fluorescent signals upon sensing glutamate using...addition to the original PDGFR TMD, the researchers introduce iGluSnFR3 in two additional anchoring domains ...induction to express soluble protein CAG: Generic strong promoter. Formulated as AAV-DJ for neuronal expression...
  3. Synthetic Photobiology: Optogenetics for E. coli

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    Blog Post
    ... to neuroscience. In fact, precise light-based control of biological processes is highly useful in other... to be optimized by Tabor’s lab, making light-controlled gene expression in E. coli easier and more robust...engineered to deliver precise, consistent, and controllable pulses of light. Complicated patterns of light...new pathways with sophisticated gene expression controls. Most cell types do not respond to low-intensity...two systems can be coexpressed for sophisticated control of gene expression. An E. coli light-sensitive...from this publication at Addgene. Multichromatic control of gene expression in Escherichia coli. Tabor JJ...
  4. Antibodies 101: ChIP

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    ...need to ensure you have a bead-only control alongside your input control. Elute the DNA after washing and..., which uses formaldehyde or UV light to create strong but reversible bonds. This will keep the protein...of histone modifying proteins, which bind quite strongly to DNA, crosslinking may not be necessary. This...Enzymatic digestion, while gentler on the sample, introduces bias due to the selective nature of the cutting...Antibodies 101: Polyclonal Antibodies Antibodies 101: Introduction to Antibodies Resources on Addgene.org ...
  5. Multiplex Genome Editing with CRISPR-Cpf1

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    ...sequence NGG TTTV PAM site preserved? Usually Destroyed Yes, Cpf1 cleaves 5' of the protospacer Multiplexing...Cassettes transfection gRNAs expressed from a polycistronic transcript  Requires co-transfection of Cas9...Cassettes transfection gRNAs expressed from a polycistronic transcript Requires co-transfection of Cas9  ...showing that Cpf1 can cleave an array of 4 crRNAs in vitro and when expressed in 293 cells. This allows for...a GFP tag. Four weeks post-infection, there was strong expression of Cpf1 and GFP in the targeted brain...effective method with multiple applications (in vitro, in vivo, transfection, viral transduction) for ...
  6. Antibodies 101: Selecting the Right Antibody

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    ...Be prepared to troubleshoot and be sure to include proper positive and negative controls as you get your...someone to turn to for advice if you need to troubleshoot. What do you know about the vendor and the vendor...anti-tag antibody; and immunoprecipitation-mass spectrometry strategies that isolate an antibody’s immune...complexes and confirm interacting partners by mass spectrometry (Uhlen, 2016). Be sure that the antibody you...The Basics of Western blottingAntibodies 101: Introduction to AntibodiesAntibodies 101: Epitope availability...
  7. Transferable Skills Guide: Problem Solving

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    Blog Post
    ...Scientific Support Team here at Addgene, so troubleshooting molecular biology experiments was in the job...core part of our academic training.  Developing strong problem solving skills is just a matter of applying...think back to the last experiment you had to troubleshoot; you probably did not just automatically come...As such, scientists are uniquely prepared to be strong problem solvers both in and out of the lab. The...position. Of course, there are limits to how far strong problem solving skills alone can take you - just...effectively does not mean I am qualified to be an astronaut. But, knowing that this skillset is a part of ...
  8. It’s Bioelectric! An Exciting Interdisciplinary Field of Research

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    ...regeneration studies, and cancer biology. I was first introduced to bioelectricity when I attended the Developmental...living cells. Since bioelectrical signals are controlled by the opening and closing of ion channel proteins...genetically encoded, light-gated ion translocators to control tumorigenesis." Oncotarget 7.15 (2016): 19575. ...signaling networks: exploiting voltage gradients for control of growth and form." Annual review of biomedical... neural and gap junction protein-mediated cues control polarity during planarian regeneration." Developmental...Vaibhav P., et al. "Transmembrane voltage potential controls embryonic eye patterning in Xenopus laevis." Development...
  9. Writing Scientific Manuscripts: Literature Searching, Reading, & Organizing

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    ...research that was done and what it means. The introduction should give you just enough information to allow... addressed. In primary research papers, the introductions should be fairly focused. Consult a review article... etc)? Did they perform all of the appropriate controls? Refer back to the methods section for finer points...automate the process as well. Sign up for eTOC (electronic table of contents) alerts from journals you read...useful papers. For many, this collection is purely electronic. Gone are the days of file cabinets filled to...She is a fan of photosynthesis and writes about autotrophs and biochemistry on her blog. Follow her on Twitter...
  10. A Look at Addgene's QC Process

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    ...Tips for Plasmid Quality Control Plasmids 101: In-house NGS Plasmid Quality Control Now at Addgene Bioinformatics... has many points at which small errors can be introduced. Eventually, these errors could cause the plasmid...Figure 1. Potential points where DNA errors can be introduced, indicated by exclamation marks. Error points...Addgene, we put a lot of effort into our quality control (QC) processes to screen for and reduce these errors...non-negotiable. By maintaining a high standard of quality control, we have built a trusted plasmid catalog containing...
  11. CRISPR in the Clinic

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    ...response (e.g., graft vs. host) as well as the introduction of the cancer-fighting component, CAR. Clinical... edit with the goal of ‘fixing’ mutations or introducing a desired edit, but this particular class of ...of treatments has quite the opposite aim: to destroy. One treatment currently in clinical trials includes...but are not limited to, ones targeting muscular dystrophy, cardiovascular disease, inflammatory diseases...by Kendall Morgan and updated in 2023 by Susanna Stroik.  References and Resources References Yuxuan Wu...blog CRISPR Antimicrobials Treating Muscular Dystrophy with CRISPR CRISPR 101: Drug Discovery and CRISPR...
  12. Viral Vectors 101: Calcium Sensors

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    ... calcium binding proteins (e.g., calmodulin or troponin C). Their fluorescent readouts are used to monitor... is a FRET-based calcium reporter derived from troponin C. It is engineered with a reduced number of calcium...fluorescent state only by simultaneous (1) user-controlled illumination with violet light and (2) exposure...different versions optimized for neuron-specific, astrocyte-specific (GCaMP6 only) or mammalian cell expression...101: Viral Applications Viral Vectors 101: An Introduction to AAV Find AAV Calcium Biosensor Vectors at... Susanna Stroik...
  13. CRISPR 101: Anti-CRISPR Proteins Switch Off CRISPR-Cas Systems

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    ...activity in HEK293 by reducing editing from ~30% in control samples to 0-10% in Acr samples. Acr proteins can...When added to human cells six hours after the introduction of Cas9 RNPs, AcrIIA4 reduced off-target editing...specific site. 2. Temporal, spatial, or conditional control of CRISPR activity Regulating Acr proteins by inducible...molecules could achieve rapid, dynamic, and spatial control of CRISPR-Cas activity. For example, fusing AcrA4II...LOV2 domain allows anti-CRISPR activity to be controlled by blue light (Bubeck et al., 2018). 3. Reduce...Your Next Experiment Read more about Optogenetic Control of CRISPR   Resources on Addgene.org Find CRISPR...
  14. Plasmid-based Recombinant Monoclonal Antibodies: What They Are and Why You Should Be Excited About Them

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    ...are monoclonal antibodies that are generated in vitro using synthetic genes that are typically expressed... a patient’s blood), scientists can use mass spectrometry to determine the amino acid sequences of the...process of antibody selection and maturation in vitro by selecting for antigen binding from libraries ...or mammalian display (Tsuruta et al., 2017). In vitro selection allows scientists to create antibodies...into an expression plasmid, the plasmid can be introduced into host cells, such as bacterial, yeast, or...on the Addgene blog Read our Antibodies 101: Introduction to Antibodies blog post Learn more about hybridomas...
  15. Open Resources and Plasmid Tools For Studying C. elegans

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    ...approach of CeNGEN, check out their publication.   3’UTRome The 3’UTRs of mRNA are important for post-transcriptional...expression in C.elegans. In this system a promoter controls the expression of a GAL-4 driver that can bind...also find additional SapTrap plasmids used for introducing point mutations that was published in a microPublication...Central PMCID: PMC3531189. Mangone, Marco, et al. "UTRome. org: a platform for 3′ UTR biology in C. elegans... L., & Jorgensen, E. M. "SapTrap vectors for introducing point mutations with unc- 119 + selection." MicroPublication...split intein for refined spatiotemporal transgene control in Caenorhabditis elegans." Proceedings of the ...
  16. Plasmids 101: Protein Expression

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    ...101 eBook! Researchers use various techniques to control protein expression for experimental, biotechnological...successful. Some mammalian systems also allow for control over when a protein is expressed through the use...Protein expression from each of these vectors is controlled by a different promoter resulting in different...to E. coli. Of all the vectors, pET, under the control of the T7 lac promoter and induced by lactose, ...researchers often place recombinant proteins under the control of the galactose inducible promoter (GAL). Other...cell-free expression systems, proteins are assembled in vitro using purified components of the transcription and...
  17. CrispyCrunch: High-throughput Design and Analysis of CRISPR+HDR Experiments

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    ...donor templates and sequencing primers for quality control. At such a scale, automation is crucial, both for... to avoid re-cutting when needed. It gives you control over your data. CrispyCrunch keeps a record of ...repair outcomes, which is important for quality control. The only ones we found that worked with NGS data... 0 and 100 for each guide, with 100 being the strongest interaction between the guide and the target and...interactions (e.g., scores <20) are reduced to 0 and strong interactions (e.g., scores >80) are transformed...weak gRNAs (CFD < 20) bind almost not at all, and strong gRNAs (CFD > 80) bind almost as much as perfect...
  18. Fluorescent Proteins 101: Fluorescent Protein Timers

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    ...placing FP timer DsRed-E5 under the control of Neurog3 (a gene controlling pancreatic differentiation) in ... (500 to 580 nm) over the course of 18 hours in vitro (see figure 1B in Terskikh et al.) and even displays...showed that their versatile timer could be used in vitro, in mammalian cells, in C. elegans, and in Xenopus...synthesized or long-lived transcription factors control gross changes in gene expression as new body sections...Pratik, et al. "A programmable synthetic lineage-control network that differentiates human IPSCs into glucose-sensitive...
  19. Tips for Using BLAST to Verify Plasmids

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    ...enjoy seeing how our quality control process has changed with the introduction of next generation sequencing...sequencing results we analyze as part of our quality control process has steadily grown. On a busy week, we ... more than 200 plasmids as part of our quality control process. Consequently our team has refined our ...database sequence is ≥ 95%. [Our QC process would be trouble-free and much faster if 95% of our results were...Plasmids 101: An Inside Look at NGS Plasmid Quality Control Learn about our Snapgene-powered plasmid maps. ...
  20. Welcome to the Lab Bench! New Protocol Series Helps Trainees Get Started in the Lab

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    ...members of the lab or those taking introductory lab courses. The Intro to the Lab Bench series is meant...supplement your teaching? You’re in luck! Addgene’s Intro to the Lab Bench series was born from the shift ...meant to be an informative introduction for those who are just starting their careers in the lab and even... need a refresher on some best practices for introductory science techniques. It’s also great for teachers...
  21. Deep Dive: Fixing and Permeabilizing for Immunofluorescence

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    ...A few months ago, we shared an introduction to immunofluorescence (IF) - a common method for visualizing...of interest within a cell or tissue. In that introduction, we broke down the method into six general steps...improve antibody binding for some targets, they can destroy it for others. That said, organic solvents’ impact... K, Mareninov S, Diaz MFP, Yong WH (2018) An Introduction to Performing Immunofluorescence Staining. Methods...resources on the Addgene blog: Antibodies 101: Introduction to Immunofluorescence Antibodies 101: Epitope...Epitope Availability Antibodies 101: Introduction to Antibodies Resources on Addgene.org Find recombinant...
  22. AAVs CREATed for Gene Delivery to the CNS and PNS

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    ...capsids (i.e. the part of the virus that determines tropism) have been developed that increase transduction...expressing plasmids is generated. Diversity is introduced into the cap plasmid library by inserting short...which, in the case of Chan et al, was neurons and astrocytes. Why screen for IV deliverable AAVs? Typically... AAV vectors expressing RGB proteins under the control of an inducer, i.e. tet-off transactivator (tTA...expression in the CNS and PNS Previously to broadly introduce a transgene into a specific cell type in the brain...fluorescent tag-then-express system that allows for controlled induction of multi-color labeling of neurons ...
  23. Lentiviral Vector Uses and Overview

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    ...researchers developed viral vector systems based on retroviruses like Moloney murine leukemia virus (MMLV). The...system that could do both, Addgene depositor Didier Trono and collaborators turned to the lentivirus HIV-1...plasmid contained a viral envelope to dictate the tropism, or types of cells the vector could infect. Finally...vectors are incredibly popular in the lab - the Trono lab’s lentiviral packaging and envelope plasmids...Gallay P, Ory D, Mulligan R, Gage FH, Verma IM, Trono D. Science. 1996 Apr 12;272(5259):263-7. PubMed ...Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, Naldini L. J Virol. 1998 Nov;72(11):8463-71. ...
  24. CRISPR Antimicrobials

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    ...resistance is swift, as few as five years after introduction. Current antibiotic strategies are nonspecific.... Phage therapy fell out of fashion with the introduction of antibiotics, but the field is currently undergoing...antibiotic-resistant bacteria (Figure 2). The first phage introduced is lysogenic, integrating into the bacterial ...lytic phage-resistant. When the lytic phage is introduced, it targets only the bacteria that are antibiotic...gain resistance to a lytic phage through the introduction of a crRNA (shown in blue). Infection with the...Bacterial Genome Engineering Treating Muscular Dystrophy with CRISPR Gene Editing Using CRISPR to Edit ...
  25. QC Sequencing Technologies at Addgene

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    ...size using electrophoresis, typically capillary electrophoresis. Capillary electrophoresis reads a fluorescent...process, see our blog post on NGS plasmid quality control. NGS is generally cheaper if you have many samples...to sequence our packaged viral DNA for quality control. This gives us a faster turnaround time than short-read...Validation: seqWell Addgene’s Tips for Plasmid Quality Control Additional Resources on addgene.org Molecular...
  26. Antibodies 101: The Four ELISAs and When to Use Them

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    ... it is essential to include proper controls. As a positive control, include a sample that is known to ...This method also requires more optimization and controls when developing, as secondary antibodies can also..., purified protein, or a peptide. The positive control will confirm that the procedure is working and ...and should always be included.  For a negative control, include a sample that does not contain the target...where the target is not expressed. The negative control will help you determine if there is non-specific...
  27. Hot Plasmids - November 2023

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    ...New compact, efficient prime editors By Susanna Stroik The Liu lab recently used phage-assisted continuous...limitations and enable impressive editing efficiencies in vitro and in vivo. Incorporating reverse transcriptase... vectors, CreVLPs, and eVLPs for in vivo and in vitro applications.   Figure 2: Modular features... enzyme, Lee and Cheah et al. achieved precise control over when and where the enzyme is active, drastically..., then the other, a dual-enrichment and mass spectrometry workflow identifies proteins bearing both labels...light-regulated LOV-Turbo enables precise spatiotemporal control of proximity labeling in living cells. Nature Methods...
  28. Hot Plasmids - August 2020

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    ...! The team used other molecular techniques to introduce time modulation of the TEV cleavage and improve...dCas-KRAB activator and the dCas-VPR repressor are introduced into the hPSC genome using AAV1 and contain an...Find the plasmids at Addgene! CRISPR RNP electroporation and AAV donor infection (CRISPR-READI) enables...new tool for synthetic biology uses CRISPRi to control gene expression. This includes a CRISPRi-based ...affinity), as well as GRAB_NEmut as a negative control. Find the sensors at Addgene! New yellow fluorescence...AAV5 and AAVrg. Find these AAVs at Addgene New controls, biosensor, chemogenetic and optogenetic AAV for...
  29. Beyond the Blood Brain Barrier: PHP.B Enters the Central Nervous System Via the LY6A Receptor

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    ...unable to reach all regions of the CNS. PHP.B, a neurotropic variant of AAV9, is better than AAV9 at gaining...receptor used by PHP.B viruses. Creating PHP.B, a neurotropic AAV9 variant PHP.B’s specificity for C57BL/6J...CNS transduction and transduces the majority of astrocytes and neurons across multiple CNS regions. PHP....Ly6a seemed the most likely candidate due to its strong linkage to the PHP.B phenotype and its expression...mice strain specific Ly6a SNPs on PHP.B binding in vitro. They found that when LY6A contained C57BL/6J SNPs...PMID: 30819613. Hordeaux, Juliette, et al. "The neurotropic properties of AAV-PHP. B are limited to C57BL...
  30. The Challenges of Cell Culture

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    ...most as cancer cells are amenable to culture in vitro. Virologists too have benefited from the ability... ‘Master’ stock, which can be stored in liquid nitrogen long term, preferably some of these vials will...will be stored in a separate backup liquid nitrogen store to prevent loss from equipment malfunctions. One...also monitor the stock levels you have in liquid nitrogen for your Working and Master stocks of each cell... laboratories to carry our independent quality control for a range of testing, from animal doping to forensics...Thermofisher Guidelines for Maintaining Cultured Cells Invitrogen Cell Culture Basics Guide Abcam Cell Culture ...
  31. Plasmids 101: E. coli Strains for Protein Expression

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    ...are available to confer greater transcriptional control, assist with proper protein folding, and deal with...Contains mutated lac permease whch allows for linear control of expression F- ompT lon hsdSB (rB- mB-) gal ...polymerase (RNAP) from the bacterial genome under control of the lac repressor, which is inducible by the...cases, some strains carry an additional measure of control such as the pLys plasmid, which suppresses basal...difference being that the latter provides tighter control of basal expression. What if I don't see protein...sources, so fear not—there are a few simple troubleshooting measures that can help get you back on track...
  32. Enabling high school research at the Journal of Emerging Investigators

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    ...Emerging Investigators Podcast breakdown Intro Chapter 1 - Introduction to Mark Springel, Olivia Ho-Shing, ... In this episode of the Addgene Podcast, we introduce you to the Journal of Emerging Investigators, an...developing one-on-one mentorship programs. Chapter 6 and Outro - Interview with high school student researchers...basic plasmid biology Use or CRISPR 101 eBook to introduce students to this amazing genome enegineering technology...
  33. New Podcast Segment: Hot Plasmids

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    ...Yeast Prototrophy Kit from Markus Ralser's Lab -  Use this kit to complement unused auxotrophies in Saccharomyces...to a quick (~5 min) Hot Plasmids podcast that introduces you to 4 new plasmid technologies from one of... Plasmid 2: Magneto 2.0 form Ali Guler's Lab - Control neuronal activity with a magnetic field. Listen...strains.  Listen to Our Segment on The Yeast Prototrophy Kit Special thanks to our sound engineer/producer...
  34. Adeno-associated Viruses (AAVs) for Genome Editing

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    ..., such as the introduction of heterozygous endogenous epitope tags, or the introduction of dominant mutations... examples from their lab include using AAV to introduce epitope tags into the endogenous alleles of the...2008). They have also used AAV gene editing to introduce naturally occurring cancer-causing mutations into...However, while CRISPR is remarkably effective for introducing non-sequence specific frameshift mutations through...efficiency of AAV-based gene editing via the introduction of a double strand break by using a targeted...
  35. Deep Mutational Scanning with One Pot Saturation Mutagenesis

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    ...coverage, mutational efficiency, control over where mutations are introduced, and scalability (see Supplementary...favoring of certain types of mutations, difficulty controlling the rate of mutagenesis, and low throughput. ...mutagenesis PCR that uses mutagenic primers to introduce all desired codon changes (step 1 in the image... high-fidelity Phusion polymerase are used to introduce point mutations into the newly synthesized DNA...Error-Prone PCR”. Methods in Molecular Biology In Vitro Mutagenesis Protocols, pg. 103-109. PubMed PMID:...
  36. Multiple Plasmids at a Low Price: Kits from Addgene

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    ...Shine-Delgarno sequences used in this so-called bicistronic design (BCD). The 49 vectors in this kit can ...of varying strengths centered within GFP/RFP bicistronic reporters characterized in Cambray et al., 2013...terminators can be used to precisely tune your polycistronic E. coli expression constructs. Some applications... Living Bacteria Using an Orthogonal Ribosome-Controlled Fluorescent Reporter."ACS chemical biology 11.1...vector that enables protein expression in any in vitro translation system. The gene set was carefully chosen...been used to produced recombinant proteins for in-vitro assays: Koch, Daniel, et al. "A pull-down procedure...
  37. Evolution of Lab Techniques

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    ... the strength of the signal in comparison to a control sample. Southern blotting applications range from...causes sickle cell anemia. The mutation happens to destroy a sequence recognized by the restriction enzyme...gels and next-generation sequencing techniques Introduced in 1977, Sanger sequencing, also known as chain-terminating...referred to as sequence DNA as it synthesized by electronically recording fluorescence activity as fluorescently...differences in ion flow caused by the different electrochemical properties of nucleotides (8). Compared to ...sequences among DNA fragments separated by gel electrophoresis." Journal of molecular biology98.3 (1975): ...
  38. Save Time and Money by Making Your Own Competent Cells

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    ... competent cells (protocol) Preparation of electrocompetent cells (protocol) At Addgene, we use the...competent cells (protocol)  Transformation of electrocompetent cells (protocol)  Cells prepared using ...with ampicillin resistance.  After a plasmid is introduced to bacteria, the bacteria is typically grown ...competent cells, don’t forget to do a negative control (plate your cells without adding plasmid) to make...have contamination.  Also conduct a positive control (test a plasmid that transforms well in other competent...
  39. Exploring Applications of the Bioluminescent HiBiT Tag

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    ...substrate generates a proportionate luminescent signal, strong enough to detect from proteins expressed at their...endogenous levels. HiBiT is compatible with both strong and weak promoters, and even modest endogenous ...quickly iterate, work across cell models, and even introduce mutations. This vector-based use of HiBiT provides...Proteins involved in endoplasmic reticulum quality control navigate a large and interconnected system of dynamic...the sensitivity of HiBiT with Addgene's quality control, support, and distribution capabilities. Give HiBiT...hepatocellular carcinoma in human tissue and in vitro. Oncology Reports, 50(5). https://doi.org/10.3892...
  40. Quick, Versatile Plant Transgenesis with GreenGate Plasmids

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    ...A few years ago, researchers introduced what’s been called a major breakthrough in cloning technology...plant model systems. “The cloning is all done in vitro; it’s no different in mouse, human or plants,” explained...of an advanced molecular plant science lab.” Introducing GreenGate As Lohmann and his colleagues describe...describe in the PLoS ONE paper that introduced the kit in December, GreenGate includes the six most frequently... available through Addgene,” Lohmann said. “We strongly believe the free sharing of material is absolutely...
  41. A new platform for fast functional characterization of potentially cancerous mutations

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    ... in a pooled cell population, as mutations with strong phenotypic effects can mask weaker ones. Thus, ... the need for a faster and higher-throughput in vitro approach. Using data from The Cancer Genome Atlas...them had not yet been functionally validated. In vitro assay allows first-time functional annotation of...collection to screen individual perturbations in vitro using Ba/F3 and MCF10A cellular models. Ba/F3 and...effect on cell viability compared to a wild-type control. Approximately ~22% of the mutations, however, ...
  42. Six Spooky Science Stories and Halloween at Addgene

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    ...and did their best to claim the costume contest trophy. We’ve had teams dressed up as plasmids, Blugene...unilateralis is a fungus that infects ants and controls their behavior. These ants become “zombies” and...their jaws into a leaf, and dies as its muscles atrophy. After the ant’s death, the fungal fruiting bodies...display imitating a moving caterpillar. The worm controls the snail’s behavior, forcing it out of hiding...blood out. Once the blood is depleted, the tongue atrophies and falls off. The louse remains becoming the ...
  43. Supporting Rare Disease Research with BeHeard 2017

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    ...afflictions. Elia Di Schiavi - Spinal muscular atrophy Dr. Elia Di Schiavi is a Research Scientist at ...Naples, Italy. His group works on spinal muscular atrophy, a disease in which disruption of the smn1 gene...the smn1 gene is known to cause spinal muscular atrophy, the molecular mechanisms behind the disease are...restore motor neuron function in spinal muscular atrophy patients. Noushin Nabavi - Peritoneal Mesothelioma...of incidence and prevalence of spinal muscular atrophy (SMA)." European Journal of Paediatric Neurology...
  44. Sequencing Options for CRISPR Genotyping

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    ... The results are then visualized using gel electrophoresis. Surveyor™ and T7E1 have been widely adopted...research setting, but these events could have disastrous health effects in the clinic. NGS-based methods...including Digenome-seq (Kim et al. 2015) for in vitro analysis, IDLV for in vivo detection (Gabriel et...potentially toxic) Digenome Seq 0.1% Cell-free (in vitro) Works across all cell types Must be verified with... background BLESS Not reported Cell-based (in vitro) Can be used on tissue from whole animal models,...most investigators use single cell clones for in vitro CRISPR experiments. The likelihood that a single...
  45. CRISPR 101: Drug discovery and CRISPR

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    .... Layers of control have also been added to the CRISPR technology to allow time-controlled edits to occur...target can be identified if it isn’t known. If a strong candidate emerges, downstream optimization is performed...generation of knockout cell lines which has made this control step extremely accessible. For example, let’s say...  Susanna Stroik...
  46. Plasmids 101: Gene, enhancer, and promoter trapping

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    ...traps can be used as high-throughput screens to introduce reporters throughout an organism’s genome. These...Enhancer traps do not need to be integrated within an intron or exon of a gene, and thus they do not disrupt...splice acceptors. They require insertion into an intron of a gene, where they can be spliced into the gene...Additional resources on Addgene.org Vector database Intro to the Lab Bench Plasmids 101 e-book Additional... Susanna Stroik...
  47. CUT&RUN: An Improved Method for Studying Protein-DNA Interactions

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    ...of input material, cells or tissue, to produce a strong enough signal over background noise. This is not...access to transcriptional regulatory elements is controlled during development using the Drosophila wing ...the target protein. This targeted digestion is controlled by the addition of calcium, which MNase requires...only for a short period of time, thus precisely controlling the amount of cutting and mitigating noise generated...protocol, the most significant being the usage of a strong detergent to permeabilize cells rather than to ...
  48. Networking and Inspiring Young Scientists at Freshers Fairs

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    ...ways budding young scholars across Europe get introduced to the cultures and opportunities available to... societies to promote their upcoming events and intro sessions, and give out useful items and information...fairs are also where many future scientists are introduced to the biology and chemistry societies that will... to attend. We thought we’d take some time to introduce you to the idea of the Freshers Fair and some ...Scientific societies like those mentioned above provide strong foundations upon which young scientists can build...
  49. Viral Vectors 101: Transductions

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    ...are similar to transfections, processes which introduce foreign nucleic acid into cells by any means, ...safety considerations, they aren't dangerous for controlled lab usage. Using viral transductions can offer...selected to perform the transduction with: either retrovirus, lentivirus, adenovirus, or AAV. Then, your desired...integrate into the host genome (lentivirus and retrovirus) while others are non-integrating (adenovirus... Susanna Stroik...
  50. Three Tips for Preventing Viral Plasmid Recombination in Your Samples

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    ...recombination of bacterial plasmid DNA: electron microscopic analysis of in vitro intramolecular recombination. ...One of the most frequent problems that I help troubleshoot involves viral vectors and plasmids with repeating...testing your prep can save you the headache of troubleshooting failed experiments. Pick the right strain While...backbone.   Ready, set, grow! Our 5 Tips for Troubleshooting Viral Transductions will help with the next...
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