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We narrowed to 1,681 results for: ina

Showing: 441 - 460 of 1681 results

  1. Hot Plasmids: Fall 2024

    Type
    Blog Post
    ...Addgene. Anti-V5 [IPI-SV5-Pk1] is a recombinant version of the original V5 tag mouse monoclonal antibody,...different antibody-based applications.  IPI’s recombinant version is a fusion of mouse variable and rabbit...with the multiple myeloma drug mezigdomide and ubiquitination substrate Ikaros (IKZF1ZF2) (B). Image adapted...structure-guided drug design and studies of protein ubiquitination and degradation.   Find the CRBNmidi plasmid...4 protein, mScarlet3 was 50% brighter than the original worm-optimized wrmScarlet. mScarlet-I3, while ...104 times higher than traditional homologous recombination methods). By using mCherry for selection of ...

  2. Plasmids 101: Stringent Regulation of Replication

    Type
    Blog Post
    ...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...

  3. Plasmids 101: Broad Host Range Plasmids

    Type
    Blog Post
    ...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...

  4. Antibodies 101: Immunoprecipitation

    Type
    Blog Post
    ..., Bjorck 1984). Protein A, Protein G, or the recombinant Protein A/G are conjugated to agarose or magnetic...their primary antibody. To circumvent this, a recombinant protein, Protein A/G, was developed, which can...primary antibody and serum immunoglobulins indiscriminately, causing competition for binding sites. In...antibody will not co-elute with the target protein, eliminating the risk of the primary antibody interfering ...antibodies conjugated within the pores are often inaccessible to their targets. Magnetic beads are solid spheres...need to be verified using alternative methods.  Finally, IP can also be used to study proteins that interact...on Addgene.org: Find plasmids encoding recombinant antibodies at Addgene  ...

  5. Four Base Editing Reporters to Monitor and Enrich Editing in Real-time

    Type
    Blog Post
    ...three codons in eGFP to eliminate fluorescence. This created three inactivated eGFP reporters- eGFP L202...reporter that monitors APOBEC (a family of cytidine deaminase proteins) and Cas9-mediated editing in real time...downstream constitutively active eGFP. To create the inactive mCherry, the lab introduced a 43 base-pair insertion...conversion to GFP after being targeted by a cytidine deaminase base DNA editor. Specifically the BFP mutant (... Figure 3: Targeting pEF-BFP with a cytidine deaminase base editor results in shift in emission spectra...resistance markers, and other enzymes such as Cre-recombinase drastically expanding the base-editing reporter...

  6. Finding nucleic acids with SHERLOCK and DETECTR

    Type
    Blog Post
    ...Originally published Aug 30, 2018 and updated April 16, 2020. Sensitive and specific nucleic acid detection... diagnose the presence of SARS-CoV-2 RNA, but inadequate access to reagents and equipment has become a... from a sample is first amplified using RPA (recombinase polymerase amplification) or reverse transcriptase...detection by Cas13. This amplification step in combination with the ssRNA reporter enables SHERLOCK to detect..., Cox DBT, Shmakov S, Makarova KS, Semenova E, Minakhin L, Severinov K, Regev A, Lander ES, Koonin EV,...2018) CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science 360...Makarova KS, Wolf YI, Gootenberg JS, Semenova E, Minakhin L, Joung J, Konermann S, Severinov K, Zhang F,...

  7. Changing Labor Laws Bring Increased Postdoc Wages

    Type
    Blog Post
    ...not. The lack of an exemption for postdocs in the final ruling was, for a lot of us, frankly quite surprising...which leads to many complications, including the inability of some institutions to know exactly how many ...of 10.4%. The Icahn School of Medicine at Mount Sinai in New York is raising the minimum from $43,692 ... funding climate combined with the lack of a sustainable research enterprise, there is no easy way for...cheap trainee workforce has clearly not been a sustainable approach to biomedical research in the U.S. and...we must make real changes to make the system sustainable. For years, there have been recommendations that... they are necessary to produce a healthy and sustainable research ecosystem and that we cannot ignore ...

  8. Podcast: Interview with Addgene's Founders

    Type
    Blog Post
    ...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 ...

  9. Technical Design of a Western Blot

    Type
    Blog Post
    ...validation data, or follow the references to find the original publication and ensure that it includes validation...-lot. The highest reproducibility comes from recombinant antibodies (rAbs), which are produced from plasmids...plasmid sequencing. While rAbs are monoclonal, recombinant multiclonal antibodies — mixtures of different...re-purchasing your primary antibody with the new reporter. Finally, each new conjugate will have to be tested to ensure...It is recommended for small to medium proteins. Finally, there are dry/electroblot transfer systems available...range, they cannot be read with equal accuracy. Finally, they degrade quickly and need to be read fairly...compatible with stripping and probing.   Imaging At the final step of your western, you’ll have two choices: x-ray...

  10. CRISPR 101: Targeting RNA with Cas13a (C2c2)

    Type
    Blog Post
    ...Cas13a: An RNA cleaving CRISPR nuclease Cas13a was originally identified in 2015 (Shmakov et al., 2015). They...enzymatically “active” state rather than reverting to an inactive state like Cas9 or Cpf1. As a result, Cas13a will...sequence of interest (red) is amplified using Recombinase Polymerase Amplification (RPA) or Reverse-transcription...crRNA targeting the sequence of interest and an inactivated fluorescent RNA reporter. If the target sequence... whether the target sequence is present in the original pool of nucleotides.   What are the potential...regulating expression of specifically targeted RNAs. Finally (and perhaps most interesting), Cas13a’s propensity...-targets. They subsequently used catalytically inactive dLwaCas13a as a programmable RNA-binding protein...

  11. Fluorescent Proteins 101: Aptamer Fluorophores

    Type
    Blog Post
    ...presence of that metabolite (Fig.2). Spinach eventually evolved into Spinach2, a more stable and brighter version...essentially mimicking GFP.  Though the aptamer, called Spinach, required longer exposure time than its protein...possibilities. For instance, further selection of Spinach with a target metabolite creates an aptamer that...but it still had limitations in vivo. Using a combination of SELEX and fluorescence-activated cell sorting...also has a higher melting temperature than its Spinach ancestors, and has a lower magnesium dependence...Chemistry 27(2):108-117 · February 2008. 11. Svensen, Nina and Jaffrey, Samie R. “Fluorescent RNA Aptamers ...Activity with GFP Resources at Addgene.org Find the Spinach Plasmids at Addgene Find Broccoli Plasmids at Addgene...

  12. Getting the Most from Your Lentiviral Transduction

    Type
    Blog Post
    ...hinders experiments is mycoplasma contamination. Mycoplasma contamination has been shown to arise from a ...Mycoplasma contamination of cell cultures: Incidence, sources, effects, detection, elimination, prevention... growth and behavior. Signs of a mycoplasma contamination can be subtle and may include reduced saturation..., labs should routinely test for mycoplasma contamination. There are several commercially available mycoplasma...PCR-based approaches to detect even low levels of contamination. For those labs that prefer a more economical....C., Drexler, H.G. “Detection of mycoplasma contaminations.” Methods in Molecular Biology 946 (2013). ...

  13. Plasmids 101: E. coli Strains for Protein Expression

    Type
    Blog Post
    ...coli strains are not usually well suited for recombinant protein expression. Many challenges can arise...coli. We will review the potential pitfalls of recombinant protein expression and some of the most popular...or are enzymatically active. For this reason, recombinant proteins are typically expressed in E. coli engineered..., which reduces proteolysis of the expressed recombinant proteins. lon protease: Strains where this is...proteins. hsdSB (rB- mB-): These strains have an inactivated native restriction/methylation system. This means...plasmids utilize inducible promoters, which are 'inactive' until an inducer such as IPTG is added to the...to make protein.  The DE3 lysogen/T7 promoter combination is the most popular induction system. The DE3...

  14. Fluorescent Proteins 101: When GFP lets you down

    Type
    Blog Post
    ... 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...

  15. Harnessing Bacterial Toxins for Allelic Exchange

    Type
    Blog Post
    ...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...

  16. How to Negotiate a Successful Lab Start Up Budget

    Type
    Blog Post
    ...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...

  17. Deep Dive: qPCR

    Type
    Blog Post
    ...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...

  18. Inside Addgene's 10 Year Anniversary Gala

    Type
    Blog Post
    ...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 ...

  19. Supporting Rare Disease Research with BeHeard 2017

    Type
    Blog Post
    ...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...

  20. Three Key Considerations for Precise Neuronal Targeting Using AAV Technologies

    Type
    Blog Post
    ...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...
Showing: 441 - 460 of 1681 results