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Showing: 281 - 300 of 954 results
  1. Your Lentiviral Plasmid FAQs Answered

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    ...of the vector components are HIV-based? A7: No single plasmid contains all the components necessary to ...The components are divided as follows: Transfer vectors contain minimal cis-acting HIV components: LTRs...decreasing the probability of creating replication-competent lentivirus (RCL) in your target cells.  Q4: How...targeted. Q6: Are transfer plasmids replication competent? What is SIN? A6: Most (if not all) Addgene transfer...LTRs, PPT, RRE, and psi packaging signal. Viral components typically total <1.5kb, which is rarely more ...are: 1) the potential generation of replication competent virus [usually HIV-1]; and 2) the potential for...
  2. QC Sequencing Technologies at Addgene

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    Blog Post
    ...have the full picture of the puzzle, the QC team compares it to the plasmid information or depositor-provided...depositor-provided sequences to confirm the correct plasmid components. For more information on this process, see our...small sample sizes, small regions of interest, or complex sequences such as G-C rich areas, secondary structures... NGS technology, due to the high-throughput compatibility. However, there are some instances where we ...thousands or even millions of base pairs. Several companies have worked on this technology, but Oxford Nanopore...sequence an entire plasmid in one read. Runs can be completed in anywhere from under an hour to 72 hours, with...data output. This method is great for resolving complex and repetitive sequences, but can be costly, low-throughput...
  3. Plasmids 101: Stringent Regulation of Replication

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    Blog Post
    ...the cell must have enough nutrients available to complete the entire round of replication. Initiation is...initiate replication. But pSC101 requires a third component: RepA, a protein encoded by pSC101 itself. RepA... are efficient for the host, as no additional components are necessary. Iterons are not the only tool ...Inc, also encoded by the ColIb-P9 plasmid, is complementary to part of the sequence forming this pseudoknot...restraining further replication.   Figure 3: Comparison of replication regulation mechanisms of stringently...replication. At high copy numbers, RepA proteins form complexes between iterons on different plasmids in a process...propagation by limiting their copy number and compensating for the additional metabolic cost imposed on...
  4. Hot Plasmids - November 2023

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    Blog Post
    ...  Here's what you'll find in this post:  New compact, efficient prime editors A versatile and precise...tape Teaching an old antibody new tricks   New compact, efficient prime editors By Susanna Stroik The ....), while PE6c and PE6d were most efficient at complex edits, such as long or highly structured templates...Phage-assisted evolution and protein engineering yield compact, efficient prime editors. Cell, 186(18), 3983–4002...interchangeable set of fusogenic and targeting components to reprogram delivery vehicles like lentiviral...DIRECTED particles where the fusion and targeting components are interchangeable. Image from Strebinger et...the range of targetable cell types. DIRECTED is compatible with fusogens from different viral families and...
  5. Antibiotic Resistance: An Old Solution but a New Problem

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    Blog Post
    ...intense amount of competition between microorganisms for available resources. This competition drives microbes...antibiotic compounds. In such a case, the microbe itself would be unharmed by the compound it produces...treat infections, we are treating microbes with compounds similar to those they have been battling for centuries...treatment regiment might wipe out bacteria that are completely susceptible to a drug while a full course of ...likely to develop over time. It isn’t feasible to completely eliminate the development of resistance; it’s...hygiene standards. Handwashing is an essential component of preventing infection, as is cleaning and disinfecting...
  6. From our Table to Yours: An Inside Look at Lunch at Addgene

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    Blog Post
    ...lab. Iron Chef-style competitions Every few months, we’ll have a cooking competition featuring a selected... eat together every day, sharing ideas for the company, but also talking about sports, movies, and just..., which is either an indication of the stiff competition or says something about my cooking.   This is... to organize. We’ll dedicate a post to these competitions in the future, and hopefully include some of...To-Do list long enough that there is no hope of accomplishing it all. How do we encourage people to take a... Hack Days, and we’ve seen great success at accomplishing “we-should-really-make-time-for-that” type projects...
  7. Adeno-associated Viruses (AAVs) for Genome Editing

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    ...likely not a coincidence that the AAV genome is composed of single stranded DNA, which is particularly ... interest, ordering them from a gene synthesis company such as IDT or Genscript, and cloning them sequentially...infection with adeno-cre. AAV-based gene editing compared to CRISPR editing CRISPR, of course, has started...and virtually never exceeds 40%. While this is comparable or better than the efficiency of most current...systems. Ohmori et al 2017 use a dual vector system composed of SaCas9 and a gRNA in one AAV and a repair template...researchers have devised methods to deliver CRISPR components as ribonucleoproteins (RNPs) that can be supplied...2017 recently showed that by delivering CRISPR components as RNPs and delivering two halves of a repair...
  8. Deep Mutational Scanning with One Pot Saturation Mutagenesis

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    Blog Post
    ...based on its library frequency before selection compared to its library frequency after selection. Key ...and easy technique that can be used to generate complex libraries of mutant plasmids ready for deep mutational...libraries, they all have inherent shortcomings compared to one pot saturation mutagenesis. Error-prone...vector with Gibson assembly. While this is a comprehensive mutagenesis method, a drawback to using Gibson...scanning libraries. PFunkel: Of prior existing comprehensive mutagenesis methods, PFunkel offers the best...second universal primer is used to synthesize a complementary mutant strand (step 2 and 3). Then the uracil-labeled...PCR-based approach for generating a customizable comprehensive mutagenesis library that’s ready to be tested...
  9. Multiple Plasmids at a Low Price: Kits from Addgene

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    Blog Post
    ...now available. However, it can be difficult to compare their performance, as most translation initiation...directly engages the ribosome for translational complex assembly. PhD student Dejan Gagoski used SITS ...SITS to create the pCell-Free vectors, Gateway-compatible backbones that enable cell-free expression of proteins...-tagged human ORF clones to allow testing and comparison of different cell-free expression systems; the... construct barcoded, locus-specific libraries compatible with Illumina next generation sequencing in order...libraries using a 2-step PCR-based protocol and are compatible with single-end or paired-end read flow cells...21408-21413. PUF Assembly Kit PUF proteins are composed of repeat segments that can be engineered to bind...
  10. How to Negotiate a Successful Lab Start Up Budget

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    Blog Post
    ... Now that you’ve completed a resource budget, and know how to decide which components are the most critical...an account of the material resources needed to accomplish your science, and can include: equipment, reagents...then start identifying the resources needed to accomplish that goal. 2. Identify the tools in that timeline...research success. If you can get away with using computer modeling and collaborations rather than using ... most start-up P.I.’s. It doesn’t have to be complicated, or difficult to create this list. Start BEFORE...equipment. That’s why a resource budget can help you compare the monetary and empirical value that helps you...from academia altogether and instead started a company around his own research. He was clear on his science...
  11. A Control for All Seasons

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    Blog Post
    ...Burry 2011). Complex experiments Finally, additional controls may be required for more complex experiments...expected response and have a baseline with which to compare the experimental group.     Figure 1: A...western blot several times with the same samples and compare the results across runs. If the results vary considerably...will briefly touch on.  Western blotting When comparing samples in different lanes in a western blot, ... to the controls listed above, viability and compensation controls are recommended for flow cytometry....example, propidium iodide only stains dead cells). Compensation controls address the spectral overlap that occurs... occurs during multiplexing. Compensation controls are single stained samples for each fluorophore and...
  12. Viral Vectors 101: AAV Variables That Matter

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    Blog Post
    ...Using AAV vectors in complex biological systems can be tricky at best, and downright infuriating at worst...Serotypes Serotype refers to the different protein compositions of AAV capsids, which determine how a virus ...what works great in one system or cell type can completely fail in a different system (Issa et al., 2023...designing and testing your AAV.  Figure 2: Comparison of AAV serotypes 1, 2, 5, 6, 8, and 9 in two ...Taschenberger et al., 2017).     Figure 3: Comparison of two astrocyte selective promoters demonstrating...Konishi, Y., Hosokawa, Y., & Konishi, H. (2014). A comparative analysis of constitutive promoters located in...vector-mediated transduction of the corticospinal tract: Comparison of four promoters. Gene Therapy, 28(1–2), 56–...
  13. Plasmids 101: Dimers and Multimers

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    ...reduced transformation and transfection efficiency compared to the monomeric version of the plasmid (Maucksch...multimers have additional origins of replication compared to monomers, they are also able to replicate at...bacterial cells with multimers grow more slowly compared to those containing the monomeric version of the...and more prone to being lost in bacterial cells compared to monomers, although this can be prevented via...cannot be used to detect plasmid multimers. Some companies provide NGS whole plasmid sequencing services ...frequency of multimerization, and in some cases completely prevent the formation of plasmid multimers (Bedbrook...produced the highest yield of monomeric plasmid compared to DH5α. However, more research is needed to determine...
  14. Antibodies 101: The Basics of Western Blotting

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    ...to detect a single, specific protein within the complex mix derived from cells or tissues. Westerns are...sample per lane, onto the top of a resolving gel composed of the crosslinked polymer acrylamide. In addition...proteins are immobilized onto a membrane, usually composed of a nitrocellulose or polyvinylidene difluoride...membrane.  In a wet transfer system the sandwich is completely submerged in a tank filled with a transfer buffer...confirming that the protein is the expected size as compared to the protein standards.  Protein abundance In...loading control when assessing protein abundance or comparing samples across lanes.  For a typical loading control...present in all samples. Uneven sample loading or incomplete protein transfer causes inconsistent levels of...
  15. Plasmids 101: Repressible Promoters

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    ...binary systems. In recent years, a number of more complex systems have also been developed which don’t fit... Bujard, both the promoter and transactivator components of the system have been engineered and improved...revolutionized genetic studies in flies. An additional component, the GAL80 repressor, adds a repressible element...UAS-driven gene expression. To further increase the complexity of the system, researchers have used split GAL4... developed by Lai and Lee. LexA/lexAop is a complementary system to GAL4/UAS that functions in essentially... cells and ~24,000-fold in human HeLa cells as compared to basal expression. Co-expression of repressor...because it allows them to be used together for complex genetic analysis in Drosophila or for precise control...
  16. Hot Plasmids: Fall 2025

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    ...microenvironment, immobilizing the components and altering the composition and dynamics. The researchers demonstrated...are available, but red GECIs have been limited compared to other colors. Two recent developments add to...proteins, PinkyCaMP is the first based on mScarlet. Compared to RCaMP3 and jRGECO1a, PinkyCaMP had better brightness...to condensates, disrupting their dynamics and composition. Reproduced from Zhang et al. (2025) under CC... 2025). The team used AAV9 to deliver CRISPRa components to adipose cells to activate genes involved in... students to explore cell metabolism. Plasmid components of CRISPRkit are now available through Addgene... the Qi lab's website crisprkit.org to request complete kits or find protocols and other resources. Find...
  17. Technique: Probe Phage Genomes for Host Binding Proteins

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    Blog Post
    ...some clones being outcompeted by others in liquid culture, dilute and plate the complete library of transformants...faces as it approaches a host cell surface are complex polysaccharide structures, many of which are easily... DNA from host cell nucleases (4). To further complicate matters, even when sequenced, phage genomes tend... for cell-binding abilities (6). This can be accomplished using a restriction enzyme with a short recognition...involves forcing DNA through a small opening using compressed nitrogen gas; this shears DNA at random locations...your resultant plasmid libraries into chemically competent E. coli and analyze transformants for appropriately...transformation should be performed using a highly competent strain that you can use for long-term library ...
  18. Science Career Options

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    ...technology, and good computer skills will give you an advantage. Strong writing, computer skills (Word, Excel...you can fully investigate and solve a problem or complete a project, overcoming inevitable failures and ... Nature Photographer/Artist Ecologist     Computer and writing skills Writing a thesis or dissertation...which are useful to many employers. Increasingly, computer skills are highly desired, as roles are becoming...Will you be coordinating projects? Do you need computer programming skills? Other things to consider...as forensics currently have too many applicants compared to the number of available roles). Make your resume...
  19. Choosing Your Fluorescent Proteins for Multi-Color Imaging

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    ... interested in are compatible with your microscope optics, you will want to compare the excitation and...fluorescent proteins currently available. Choosing compatible fluorescent proteins To choose a set of fluorescent...scope of this post. Are your fluorescent proteins compatible with your microscope optics? To determine if ...is efficiently collected by the microscope. To compare the match between a fluorescent protein and a filter...here's a screenshot from the Chroma Spectra Viewer comparing a standard Cy3 or Rhodamine filter set (Chroma...channels used for more abundant proteins or marking compartments. I hope this sheds some light on multicolor ...
  20. Plasmids 101: Mammalian Vectors

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    ...Jurkat T cells Complexes with DNA; causes strand scissions 100-400 ug/mL *Not comprehensive.   ** In eukaryotes...cells. Of course, these mammalian vectors must be compatible with the cell type they are tranfected into –...cells. Before getting into the mammalian plasmid components, it may be useful to describe the means of introducing...called transfection. Transfection is somewhat comparable to bacterial transformation (the introduction...void. These ORIs, however, require additional components expressed in trans within the cell for effective...ORIs, respectively. The presence of these viral components greatly reduces the rate of plasmid dilution ...
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