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We narrowed to 919 results for: IRE;

Showing: 241 - 280 of 919 results

  1. Choosing the B(right)est Fluorescent Protein: Photostability

    Type
    Blog Post
    ...photoswitching (Kremers et al, 2009), which usually are undesired properties. In the ideal situation, a fluorescent...illumination, TIRF or spinning disk confocal use entirely different excitation power regimes. Therefore,...that a photostable fluorescent protein is a key requirement for quantitative live-cell imaging, it is important...photostability of the fluorescent proteins can be directly compared. An example of a photostability measurement...proteins or localized fusion proteins and do not require dedicated plasmids or constructs. It should be ...imaging would be mTurquoise2, based on the data acquired at low excitation powers. Note, however, that,...Plasmids for Subcellular Localization Browse the entire Fluorescent Protein Collection ...

  2. Developing Lab Management Software for Biology

    Type
    Blog Post
    ...failures require re-attempting the same step, some require going back two steps, some require starting... our insights can help steer you in the right direction when writing your own software. None of this is...starting over entirely, and our software needs to handle these cases. The following code examples show how software... software simply follows a linear progression directing our techs through the process. The problem? As... itself. If something happens to the worm that requires a change of tube, there’s no clean way to separate...

  3. Hassle-free 96-well Format Epitope Tagging Using Cas9 Ribonucleoprotein

    Type
    Blog Post
    ...challenge as it relies on inefficient homology-directed repair (HDR) to correct double-strand breaks (...tagging, however, suffer from many pitfalls: they require construction of plasmid vectors and/or rely on ... lines, especially for ambitious projects that require epitope tagging of a large cohort of genes (e.g...cells in the field (DAPI). Using the ICC assay as a direct readout of tag insertion efficiency, we have recently...also straightforward as the RNA molecules can be directly purchased and there is no need to construct plasmid...more effectively than Cas9 complexed with sgRNA. Direct comparison of Cas9 RNPs prepared with an in vitro-transcribed...was possible because reagents could be purchased directly from commercial suppliers without investing time...

  4. Engaging with science and society at pgEd

    Type
    Blog Post
    ...government; advising writers and directors in Hollywood; and working directly with community organizations...This guest post was contributed by Johnny Kung, Director of New Initiatives for the Personal Genetics Education...dialogue between scientists and communities. As the director of new initiatives at pgEd, I take on a variety... beyond the biological sciences, as well as to acquire leadership and communication experience. These ...communication A career in science communication or policy requires broad interest in issues at the interface of science...implications of their work. Johnny Kung is the Director of New Initiatives for the Personal Genetics Education...

  5. Educational Planarian Tools for Teaching Developmental Biology Techniques

    Type
    Blog Post
    ...multi-cloning site flanked by T7 promoters to allow for bidirectional transcription to create dsRNA. Students can ...Sánchez lab describes the plasmids and protocols required for both WISH and RNAi experiments, as well as... molecular cloning to insert the cDNA into the desired plasmid backbone. The Sánchez lab plasmids allow... result in  the labeling specific cell types or entire organs in planarians. For example, the gene Piwi...proteins remove one strand of the siRNA and are directed by complementarity to the remaining single stranded...knockdown of beta-catenin. Other gene targets do not require amputation to be visible. For example, odf2 gene...Link: http://abt.ucpress.edu/content/79/3/208 2. Fire, S. Xu, M.K. Montgomery, S.A. Kostas, S.E. Driver...

  6. Adeno Associated Virus (AAV) for Cell and Gene Therapy

    Type
    Blog Post
    ...However, many diseases require techniques that can deliver gene therapies directly to the cells that need...reconstitution with a normally functioning T cell repertoire (1). In this example, it was possible to transduce...genome. AAV belongs to the family Parvoviridae and requires co-infection of helper viruses like adenovirus...facilitate and enhance DNA repair via homology directed repair (HDR). This characteristic makes AAV vectors.../vectors.php). These clinical trials have been directed at diseases including but not limited to hemophilia...overcome this issue by developing new capsids through direct evolution, capsid shuffling, and peptide displays...

  7. Plasmids 101: Terminators and PolyA signals

    Type
    Blog Post
    ...the gene to be transcribed, and typically occur directly after any 3’ regulatory elements, such as the ...transcription of the template and initiating the desired cleavage event, although some engineered terminators...downstream elements. A high affinity terminator may be desired for multi-cistronic constructs where high termination...certain RNAs. Unlike the eukaryotic mechanism which requires a consensus sequence for the addition of a poly...different than Polymerases I and II, which both require binding of termination factors. Although both are...The conserved eukaryotic polyadenylation signal directs cleavage at the cleave signal and addition of a...Position-dependent sequence elements downstream of AAUAAA are required for efficient rabbit beta-globin mRNA 3' end formation...

  8. Plasmids 101: Protein Expression

    Type
    Blog Post
    ... for the expression of mammalian proteins that require multiple post-translation modifications for proper... Inducible promoters are extremely useful if a desired protein product is toxic to cells at high concentrations...their advantages, mammalian expressions systems do require demanding cell culture conditions compared to other... be quite time consuming and that insect cells require demanding culture conditions similar to mammalian...expression from each vector; lower expression may be required if your protein is toxic to E. coli. Of all the...share many of the same features and processing requirements as mammalian cell expression systems, including...allows for easier access and purification of a desired protein. Despite being a rather nascent technology...

  9. Truncated gRNAs for Regulating Gene Expression

    Type
    Blog Post
    ...researchers at the Wyss Institute for Biologically Inspired Engineering. CRISPR/Cas9 technology has revolutionized...edits. Cas9 is a DNA binding protein that can be directed to virtually any genetic locus when complexed ...exploited to either inactivate a gene or introduce a desired genetic alteration. Listen to Our Podcast Interview...and bind to DNA; when complexed with a gRNA that directs them to the promoter of a gene of interest, they...that allows users to decide which Cas9 protein is directed to which target site (1). Although this is possible...to the Cas9-gRNA complex. When this complex is directed to a target upstream of the transcriptional start...DNA but is no longer competent to cut DNA. When directed to a site downstream of the transcriptional start...

  10. The AAVantages of AAV in CRISPR Screens

    Type
    Blog Post
    ...handled at BSL-1, while lentivirus requires a BSL-2 setup. This requires a less involved setup when working...make them an attractive option. However, safety requirements and expression limitations may be too constraining...Created with BioRender.com. The in vitro option requires a bit less work for the first two steps, as no... them into mice. For in vivo screens, mice are directly infected with the CRISPR library. In vivo screens...still beats lentivirus. In mice, AAV is almost entirely non-immunogenic — meaning when conducting in vivo...Aryee, M. J., Joung, J. K., Breakefield, X. O., Maguire, C. A., & György, B. (2019). High levels of AAV...

  11. Production of Virus in Insect Versus Mammalian Cells

    Type
    Blog Post
    ...preps, lentiviral titration requires an additional couple of days, as it requires transduction of more cells...companies. Production of both AAV and lentivirus requires three plasmids — a transfer plasmid containing...available. Unlike mammalian cells, they do not require serum or an incubator to grow. The virus Insect...lentiviruses can be produced in insect cells, but they require cloning specific AAV or lentiviral machinery into...baculovirus plasmid. Production of baculovirus requires only two plasmids — your transfer plasmid and ...are readily available, so the amount of cloning required is about the same between insect and mammalian...

  12. CRISPR 101: Cytosine Transversion Editors

    Type
    Blog Post
    ...other 8 conversions are base transversions: they require switching between a two-ring purine and one-ring...the R-loop structure. In order to bind DNA, Cas9 requires (1) a guide RNA, (2) a complementary DNA sequence...DNA target strand, leaving the non-target strand unpaired and accessible on the outside of the enzyme. ...transversion editors. Red arrows indicate a conversion directly catalyzed by the base editor. Black arrows indicate...hypothesized that the CGBE edit process occurs entirely while Cas9 is bound, keeping the two DNA strands...maybe because repairing a GBE-derived abasic site requires more cellular factors binding to DNA. However,...regions that were already accessible. It's not entirely clear why this is, but it might point to a more...

  13. Getting to Know Expansion Microscopy

    Type
    Blog Post
    ...another option. However, most SRM techniques also require specialized instruments and technical expertise...material that is able to expand evenly in all directions to several times its original size. Depending...of - and ExM is no exception. First, expansion requires fixed samples. This limitation may be the both...expansion is for your sample to expand evenly in all directions, aka isotropically. But achieving isotropic expansion...optimization and careful execution, but they cannot be entirely eliminated. It is recommended that you image samples...limitations, ExM is a valuable tool, especially when paired with complementary experimental approaches. Since...demonstrated to work in different model systems, paired with SRM techniques, and adapted for detecting ...

  14. Don’t FRET: Bimolecular Fluorescence Complementation Makes Visualizing Protein-Protein Interactions Easy

    Type
    Blog Post
    ... of hard-to-achieve factors. For example, FRET requires that fusion proteins be in close proximity and...fluorescence complementation BiFC has been used to directly visualize PPIs that regulate numerous cellular...FRET, as the overexpression of fusion proteins it requires can lead to aberrant subcellular localization ... construction Fusing FP fragments to your PIPs requires careful selection of FP fragments and, preferably...where BiFC outshines FRET — BiFC experiments only require that you image and measure the fluorescence of ...information, constructing appropriate controls may requires that you empirically test a number of mutants ...is a convenient, fluorescence-based method for directly visualizing PPIs in cells. The ease with which...

  15. CRISPR 101: Making a Knock-In Cell Line

    Type
    Blog Post
    ...with tips and tricks for harnessing the homology-directed repair pathway, designing the best donor DNA, ...case for knock-ins. Knock-in mutations typically require an exact DNA sequence to be incorporated at a precise...template for repair? Read on to find out! Homology-directed repair and genome engineering The easiest way ...strand breaks – (HR). HR is the dominant homology-directed repair (HDR) pathway in mammalian cells, but a... S., Pan, H., et al. Methods favoring homology-directed repair choice in response to CRISPR/Cas9 Induced-double..., Alla, R. K., Doudna, J. A. Enhanced homology-directed human genome engineering by controlled timing ...et al. CRISPR-Cpf1 mediates efficient homology-directed repair and termperature-controlled genome editing...

  16. Bioinformatics at Addgene

    Type
    Blog Post
    ...system that we chose needed to fulfill certain requirements: Customizable to our specific needs Modern ...focus on our development instead of building the entire ecosystem Proven performance with other bioinformatics..., so our QC team could initiate and monitor the entire pipeline. Benefits of Nextflow for bioinformatics...pieces of software that depend on each other for the entire package to run. The small, portable system provided...Nextflow Establishing a complete pipeline however, required more than just identifying criteria that we needed...small with a print statement and tested that the desired data was available so we felt confident we were...process was added, we progressively constructed the entire workflow in the Nextflow script. With our completed...

  17. Hot Plasmids - November 2023

    Type
    Blog Post
    ... the creation of CAR-T cells directly within the body. Overall, DIRECTED can increase on-target delivery...unstructured templates and significantly reduced undesired edits (indels, etc.), while PE6c and PE6d were...Feng Zhang’s lab has developed a system called DIRECTED (Delivery to Intended REcipient Cells Through ...applications.   Figure 2: Modular features of DIRECTED particles where the fusion and targeting components... expanding the range of targetable cell types. DIRECTED is compatible with fusogens from different viral...experimental therapies and medical applications.  Find DIRECTED plasmids here! Strebinger, D., et al. (2023). ...

  18. Career Insights: Technical Support Specialist

    Type
    Blog Post
    ...scientific knowledge they require. Biotechnology companies vary to what extent they hire experts for their technical... to publish in high-impact journals, and the requirement for a high grant success rate. At this point,...options. While some non-academic career options require additional study for late career scientists to ... similar), there are many roles out there that require exactly what life science PhDs can offer: vast ... and Their Careers in Our Career Videos What's required for a role in technical support? It takes some...necessary. The job description and the candidate requirements (and maybe also the proposed salary range) should...

  19. CRISPR-mediated Plant Base Editors

    Type
    Blog Post
    ... only possible through exploiting the homology-directed repair (HDR) pathway which occurs in cells at ...) base. Later the resultant U-G pair is either repaired by cellular mismatch repair machinery making an...second outcome and increases the generation of the desired T-A base pair. Figure 1: Transition mutation...by base editors. Pathways that compete with the desired outcome are not pictured. Adenine base editors...not as active as uracil excision, ABE does not require any additional inhibitor protein like UGI in CBE...NGG’ PAM cannot be editable? They can, but they require other base editor platforms. Hua et al. constructed...). Type Nickase Cas9 (D10A) Deaminase PAM requirements Reference CBE SpCas9 rAPOBEC1 NGG 6 SpCas9...

  20. Changing Labor Laws Bring Increased Postdoc Wages

    Type
    Blog Post
    ...was contributed by Future of Research Executive Director, Gary McDowell. On Dec 1st, the threshold at which...when the ruling was issued, a joint blog post by Director of the NIH Francis Collins and Secretary of Labor...the institutions that are meeting the salary requirements of the FLSA. In the University of California... minima of $30,000, and raising salaries would require those postdocs receive an increase in salary of... below the exemption level. Not only is this in direct contradiction to explicit policies set up other...postdoctoral training has not been demonstrated to be required for the jobs that the majority of postdocs end...McDowell! Gary S. McDowell is currently the Executive Director of Future of Research. He is interested in advocating...

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

    Type
    Blog Post
    ...Arabinose-inducible expression of T7 RNAP; IPTG may still be required for expression F- ompT lon hsdSB (rB- mB-) gal... such as M15(pREP4) use a lac repressor to act directly on the expression plasmid in order to repress ...which can prevent the culture from reaching the desired density within a reasonable time-frame. For these...you’ve tried a common strain and don’t get the desired level (or any) protein expression? Low expression...compatible with a pLys strain. Your strain may require additional antibiotic selection or a special growth...is accomplished by growing your culture to the desired density at 37°C and reducing the temperature or...secreted to the medium or if slow expression is required due to solubility concerns. Insoluble and Secreted...

  22. Plasmids 101: The Promoter Region – Let's Go!

    Type
    Blog Post
    ...eukaryotic cell types (mammalian, yeast, plants, etc) require unique promoters and there is very little crossover...binding sites, commonly used in Drosophila Specific Requires the presence of Gal4 gene to activate promoter...Promoter from T7 bacteriophage Constitutive, but requires T7 RNA polymerase. When used for in vitro transcription... Negligible basal expression when not induced. Requires T7 RNA polymerase, which is also controlled by...Promoter from Sp6 bacteriophage Constitutive, but requires SP6 RNA polymerase. SP6 polymerase has a high ...bacteriophage lambda Can be temperature regulatable  Often paired with the temperature sensitive cI857 repressor....Promoter from T3 bacteriophage Constitutive, but requires T3 RNA polymerase When used for in vitro transcription...

  23. Evolution of Lab Techniques

    Type
    Blog Post
    ...Whereas Southern blotting is labor intensive and requires a large amount of high-quality DNA, real-time ...automation, higher throughput screening, and a lower requirement for the amount of DNA used saving the researcher...Figure 1). Though some labs may prefer to send DNA directly for sequencing (more on that below), restriction...dideoxynucleotides (ddNTPs), lacking the 3’ hydroxyl group required for the extension of a DNA strand (8). Adding ...whole genomes or profiling transcriptomes (RNAseq) require sequencing of many samples at once for later comparison...confirming the results of a cloning experiment, require only one or a few sequencing reactions and may ...sequencing technologies could one day sequence an entire genome for under $1,000 (10). The future of sequencing...

  24. Thoughts on the Future of Research 2015 Boston Symposium

    Type
    Blog Post
    ...Essentially the goal was to determine what data is required for the academic research community to develop...prepare scientists for positions outside of academia Require that postdoc salaries be paid on fellowships instead... joining long programs if it is obvious that a desired career outcome is not easily achieved. Of note...incentives that encourage PIs and universities to hire a glut of trainees at low wages will still be there...take steps to broaden their skills beyond those required at the lab bench. At Addgene we try to do our ...was the idea that simply satisfying a percent requirement for diversity in graduate students, postdocs,...individuals are discluded/not treated equitably even when hired; well-meaning mentors often fail to judge the work...

  25. CRISPR Protocol for Genomic Deletions in Mammalian Cell Lines [Video]

    Type
    Blog Post
    ...CRISPR/Cas9 for targeted genomic deletions We were inspired to produce intrachromosomal deletions based on... advent of CRISPR/Cas9, we began to explore the paired double-strand break (DSB) approach at a variety...applications in which the production of deletions by paired DSBs may be particularly useful. The first is the...context. An alternate outcome to deletions following paired DSBs is inversions [3]. These rearranged segments...loss-of-function frameshift mutations. However these may require restriction fragment length polymorphism analysis...target two exonic sites within a single gene using paired DSBs. This method allows for deletion screening...

  26. Tag Your Favorite Yeast Genes with Ease

    Type
    Blog Post
    ...protein products is desired, as multiple selection markers can ensure that all desired tags have been integrated...linear PCR product is introduced into the cell by direct transformation. A given insert will typically contain...Simply design your amplification primers with the desired targeting homology—in frame, of course—and start...ONE 8(7): e67902. Many imaging studies rely on direct fusion of fluorescent proteins (FPs) to a yeast...specific filter sets and experimental output requirements. Select from these yeast-optimized fluorophore...

  27. A Control for All Seasons

    Type
    Blog Post
    ... may be required for more complex experiments. A multiplexed experiment, for example, requires staining... negative controls Most biological experiments require positive and negative controls to ensure proper...is generally a sample or group that will have a desired response. For example, if you are studying the ...sample, the antibody binds specifically to its desired target (purple) and nonspecifically through the...The Preimmune serum control will not bind to the desired target but will bind nonspecifically through the... site.      Secondary antibody specificity  In indirect staining approaches, secondary antibody specificity...

  28. Viral Vectors 101: Optogenetic Tools

    Type
    Blog Post
    ...), and photoswitches. Light-gated ion channels directly open and close when exposed to light and change... photoswitches OptoXRs are great for activating entire signaling pathways, but what if you’re interested...potential, then a channel with fast kinetics may be required, whereas a channel with slower kinetics can be...Principles for applying optogenetic tools derived from direct comparative analysis of microbial opsins. Nature...., & Bamberg, E. (2003). Channelrhodopsin-2, a directly light-gated cation-selective membrane channel....BiPOLES is an optogenetic tool developed for bidirectional dual-color control of neurons. Nature Communications...

  29. Plasmids 101: Gene, enhancer, and promoter trapping

    Type
    Blog Post
    ...Promoter trap systems lack a promoter and thus require the trapping of an endogenous promoter. The system...traps, enhancer traps lack enhancer elements and require the trapping of endogenous enhancers to stimulate...constructs that contain splice acceptors. They require insertion into an intron of a gene, where they ...like promoter traps. While this system does not require insertion into an exon, like promoter traps, it...think enhancer traps are the top trap – they don’t require insertion in a highly specific gene region and ... the gene that they are monitoring as they are directly integrated into it. Enhancer traps do not need...

  30. Single-cell tracking of lineage and identity with CellTag

    Type
    Blog Post
    ...Direct reprogramming describes the process where differentiated cells are turned into a cell type of ...Though a valuable tool for regenerative medicine, direct reprogramming is an inefficient process, with the... the majority of cells failing to develop the desired identity. The development of single cell technologies...CellTag The Morris Lab developed CellTag to track the direct reprogramming of mouse embryonic fibroblasts (MEFs..."Single-cell mapping of lineage and identity in direct reprogramming." Nature 564.7735 (2018): 219. PubMed...pluripotent stems cells (iPSCS) and reprogramming and direct reprogramming Explore different methods for delivery...

  31. Antibodies 101: Chimeric Antibodies

    Type
    Blog Post
    ...come up with a great experimental plan that requires indirect staining of both targets in your specimen.... are the same isotype and therefore cannot be indirectly probed simultaneously on the sample. What to ...matter? Isotype matters when constraining using the indirect method since a secondary antibody reactive to ...In the case above, you can’t costain with your desired antibodies because the pair belong to the same ...the same isotype can not be used for multiplexed indirect staining methods since the secondary antibody ...

  32. Another Pathway into Cells: iTOP

    Type
    Blog Post
    ...into primary cells, so most primary cell lines require viral infection. A new paper from Niels Geijsen...they could simply induce cells to take up protein directly? It’s not a new idea, but it comes with a few ...expression of a luciferase reporter. CPP was not required for protein entry into cells! After verifying ...non-detergent sulfobetaine 201 (NDSB-201) were required for protein delivery. D’Astolfo et al. coined ...take up, and continued protein expression would require multiple transduction events. For these reasons...concerns associated with virus work. Fourth, working directly with proteins removes the limitations of packaging...

  33. The Importance of High Titer for AAV Transductions

    Type
    Blog Post
    ...are required for the transduced cell to become light responsive than fluorescent proteins required to ...always be enough and therefore higher titer will be required. For example, retrograde AAV gets transported ...with your experimental setup. Some experiments require higher gene expression and therefore higher titer...if you end up concentrating your AAV above your desired titer and want to dilute it, err on the side of...less buffer than you would to achieve the exact desired concentration to avoid over-diluting, and make ...

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

    Type
    Blog Post
    ...enabling genome-wide interrogation of the genes required for cellular processes. For a successful screen...plaques. ApoB bound lipoproteins (ApoB-LPs) are indirectly measured through their triglyceride or cholesterol...strong correlation with cardiovascular disease and indirect measurement of ApoB-LPs through triglyceride/cholesterol...et al, 2017).  In an effort to measure ApoB-LPs directly and develop a potentially high throughput in vivo...transcription. Transcription from σ54 promoters requires binding of bacterial enhancer binding proteins...difficulties such as small fluorescence changes or a requirement for very high excitation light intensities have...

  35. Choosing Your Fluorescent Proteins for Multi-Color Imaging

    Type
    Blog Post
    ...of the Nikon Imaging Center at UCSF. A common requirement for live cell imaging experiments is the ability...simultaneously. To do so with fluorescent protein labels requires multiple fluorescent proteins whose excitation... means that the choice of fluorescent proteins requires some thought. The first step in choosing fluorescent...fluorescent proteins can be separated from each other requires knowledge of their excitation and emission spectra...Beware that these infrared fluorescent proteins require biliverdin as a cofactor and so you may need to...Thorn is an associate professor at UCSF, where he directs the Nikon Imaging Center. He received his PhD in...

  36. Improved Plasmid Maps Powered by SnapGene

    Type
    Blog Post
    ...maps have the identities of most features labeled directly on them making them much easier to quickly identify... see above) for your own reference or to paste directly into your notebook. This will make it easier for... immediate, high-level analysis, you can click directly on the “Analyze Sequence” button to pull up an...sizes, colors used to indicate them on the map, direction (if relevant), and types. Types listed are standard...sequences, binding site locations, lengths, and direction. Clicking on a particular primer here also highlights...Addgene software development teams for working tirelessly to implement these improvements. Let us know ...

  37. Choosing a CRISPR Nuclease: Site Accessibility, Specificity, and Sensitivity

    Type
    Blog Post
    ...accessible by Cas9. Our previous blog post The PAM Requirement and Expanding CRISPR Beyond SpCas9 described ...a rationally engineered Cas9, SpCas9-NG, which required only the di-nucleotide PAM NG (Nishimasu et al...for base editing techniques, where Cas9 must be directed to a very specific site. Specificity vs. sensitivity...of different sites. Using a combined random and directed mutagenesis approach, the Anna Cereseto Lab created...specificity. Most recently, the Jungjoon Lee Lab used directed evolution to generate Sniper-Cas9 resulting in...Central PMCID: PMC5951633. Lee, Jungjoon K., et al. "Directed evolution of CRISPR-Cas9 to increase its specificity...

  38. GCE4All: Making Genetic Code Expansion Accessible

    Type
    Blog Post
    ... interested in using GCE, here are the basic requirements of use. First, and most obviously, you need ... GCE are essentially those needed to make site-directed mutagenesis, many researchers new to GCE aren’...field, it’s not uncommon to find protocols that require reagents which aren’t broadly or easily available...complicated.” And Ryan Mehl would know. He’s the director of GCE4All, an NIH-funded center located at Oregon...phosphoserine and, because it uses ∆serB strains, does not require exogenously added phosphoserine. A community resource...using, GCE to reach out to them and let them know directly what protocols would be useful. Think GCE could...

  39. Advice for Starting a Biotech Company from a Grad Student turned Entrepreneur

    Type
    Blog Post
    ...I like to envision that my research will have a direct impact on healthcare and the community. As a result...like-minded people, I quickly realize that a company requires more than just good data to become a product. ...settings, etc. What project in the Collins lab inspired Sample6? The initial work focused on using bacteriophages...graduate school usually) is one of the key skills required to achieve success in this business. A good presentation...expenditure of effort in preparation and practice is directly related to success presenting." A journey ...and succeeding. Take home Starting a company requires dedication, passion, and around-the-clock work...

  40. Plasmids 101: Golden Gate Cloning

    Type
    Blog Post
    ...recognition sequence, they can be customized to direct assembly of DNA fragments. When designed correctly...contains sites with complementary overhangs that direct assembly of the final ligation product. As shown... be re-digested. In contrast, formation of the desired ligation product is irreversible because this construct...a single reaction! These overhangs specify the desired order of fragments, and the loss of enzyme recognition...Exonuclease-based methods like Gibson assembly require 20-40 bp of homology at the ends of DNA fragments...cloning is not 100% sequence-independent: to avoid undesired digestion, the Type IIS site used must not be ...
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