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We narrowed to 755 results for: rest

Showing: 161 - 180 of 755 results

  1. Plasmids 101: Gibson Assembly and Other Long-Homology Based Cloning Methods

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    ...fragments, but uses site specific restriction sites (Type IIS restriction endonucleases) to cleave DNA outside...which have provided appealing alternatives to restriction enzyme cloning. These newer technologies have...They offer many advantages over the traditional restriction enzyme cloning we once relied exclusively on....that does not require the presence of specific restriction sites (or lack thereof) nor a serious time commitment..., imagine that you want to insert your gene of interest into a vector with a large tag at the N-terminus.... If you inserted these two pieces of DNA by restriction enzyme cloning, you would have to do it in two...Gibson assembly, you can insert both the gene of interest and the tag sequences into the vector in one step...

  2. Plasmids 101: Golden Gate Cloning

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    ...relies on Type IIS restriction enzymes, first discovered in 1996. Type IIS restriction enzymes are unique...If your genes of interest or destination vector contain multiple internal restriction sites that may not...Addgene’s plasmids are used with a wide variety of restriction enzyme-based cloning methods. Each method has...unique from "traditional" restriction enzymes in that they cleave outside of their recognition sequence, ... The cloning scheme is as follows: the gene of interest is designed with Type IIS sites (such as BsaI ... ligation favors formation of the construct of interest. Although efficiency may decrease with an increased...to as MoClo, this strategy uses the Type IIS restriction enzymes BsaI and BpiI/BbsI to efficiently assemble...

  3. Fluorescent Proteins 101: Visualizing Subcellular Structures & Organelles

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    ...protein of interest The first step in analyzing the localization of your protein of interest is to make...disease, researchers often isolate proteins of interest and examine them using biochemical methods thus...healthy and diseased states can also provide interesting insights into disease mechanisms and protein ...available for microscopy, by cloning your protein of interest into a vector encoding a fluorescent tag. Once...fusion, it is possible to track your protein of interest from its production site to its final destination...traffics the fluorescent protein to your organelle of interest. Click on the gene names in the figure below to...plasmids for labeling your subcellular structure of interest in the Allen Institute for Cell Science Plasmid...

  4. CRISPR 101: Validating Your Genome Edit

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    ...screen using PCR and a restriction enzyme digest.   Fig. 3: Restriction enzyme screening for resistivity...edits.   Interested in this method, but your edit doesn’t affect any endogenous restriction enzyme sites...screen hundreds of clones looking for your edit of interest. For all of the screening strategies outlined ...introduces a frameshift in the coding region of interest will get the job done. In other words, any insertion...First, use PCR to amplify the target region of interest. Then Sanger sequence the PCR product of both ... 50% are -/+, and 25% are +/+ for your gene of interest. Thus, to find a null clone, you should screen...of your experiment for a screening strategy. Restriction enzyme screening is sensitive to single base ...

  5. Quick Guide to All Things Lentivirus

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    ...express your gene of interest either constitutively or inducibly. If your gene of interest is toxic or needs...If you are interested in using lentiviral vectors to introduce your favourite gene into your favourite...and the transfer plasmid encoding the insert of interest as well as sequences necessary to encapsulate ...system was efficient at transferring the gene of interest to the appropriate cells, it was obvious that ...The ability to infect non-dividing cells is not restricted to in vitro cell culture as lentivirus-derived... optimum concentration to infect your cells of interest.   Advantages Disadvantages Can carry large...transfer plasmid is the vector you clone your gene of interest into and is an important feature for your experiment...

  6. Lambda Red: A Homologous Recombination-based Technique for Genetic Engineering

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    ...Restriction enzyme cloning is the workhorse of molecular cloning; however, one of its biggest limitations...that sequence modifications can only be made at restriction enzyme cut sites. The lambda red system is an... of DNA within E. coli and is independent of restriction sites. The lambda red system is derived from ...means cloning occurs in vivo, as compared to restriction enzyme cloning where the genetic changes occur...sections, key steps that differ from traditional restriction enzyme cloning will be explained in greater detail...Red recombineering system to replace a gene of interest with an antibiotic resistance cassette.   ...using primers that amplify the DNA sequence of interest and flank it with 50 base pairs of homology to...

  7. Multiple Plasmids at a Low Price: Kits from Addgene

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    ...with Type IIS restriction enzymes and standardized primers to clone your gene of interest into both sequencing...protein of interest. The plasmids used to generate these fusions all use the same restriction enzymes for... pExpression vectors) plasmids. The type IIS restriction sites used in these vectors are of low abundance... they are unlikely to be found in your gene of interest) and prevent incorporation of extensive sequences...kit will allow you to quickly study your gene of interest in a variety of model systems, thereby opening...downstream of which you can clone your gene of interest. Translation of the standardized leader peptide...secondary structures in the mRNA encoding your gene of interest from affecting its translation initiation and ...

  8. Plasmids 101: Screens vs. Selections

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    ...finding new factors in your biological system of interest, the so-called “fishing expedition.” Your first... process in which only cells with the trait of interest will survive in the selected growth conditions...hinder, or have no effect on the process you are interested in. What does this actually look like? For a...selected. The method you choose to assess traits of interest is highly specific to your scientific question...CRISPR, but instead of knocking out the genes of interest (or the whole genome), they knock down gene expression...RNAi libraries are especially useful if you are interested in studying genes whose knockout would be lethal...apply enough pressure to select for variants of interest, though. A selection can take the form of varied...

  9. Plasmids 101: Degron Tags

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    ...targeting or cloned in-frame to the protein of interest for plasmid-based expression systems. Degron tags...is situated between the degron and the gene of interest and the degron domain is terminal. Between the...elements (protease and degron) and the protein of interest, there is also a protease recognition site. In...the presence of the degron, and the protein of interest is untagged and functions per the usual. In the...FKBP12F36V. By fusing FKPB12F36V to your protein of interest, it can be degraded by this selective degrader...fusion protein. This will target the protein of interest to the proteasome for degradation. Like several...dimerization of an FKBP12F36V fused target protein-of-interest and the CRBN E3 ligase complex leading to ubiquitination...

  10. Plasmids 101: 5 factors to help you choose the right cloning method

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    ... on versatile Type IIS restriction enzymes. These cut outside their restriction sites allowing you to ... But...how? Cloning is no longer limited to restriction enzymes - although many labs still love using...extensively described in other articles; so if you are interested in learning more about any one of them, please...your E. coli do the work for you. Traditional restriction enzyme and ligase based cloning is a relatively...The ligase cost is comparable to that of the restriction enzymes and overall you’re looking at a price...of a few dollars per reaction for traditional restriction cloning. Sequence and Ligation Independent Cloning...appropriate inserts by PCR, there is no need for restriction digests, gel purifications, or ligations. The...

  11. Antibodies 101: Validation

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    ...protein of interest endogenously and transfect them with a plasmid encoding your protein of interest. Then ...plasmid encoding your protein of interest, known as Gene of Interest (GOI), and transfect it into your...bioinformatician) to look at RNA expression of your gene of interest across a set of human tissues.  From this data... target. Removing (or reducing) the protein of interest in your sample, should lead to reduced or eliminated...at the subcellular localization of a protein of interest. Conveniently, you found a cell line in which ...from the cells transfected with your protein of interest and show just the faintest band from the cells...on the expression of a new receptor that is of interest to you. You found a couple of antibodies that ...

  12. 28 Hot Plasmid Technologies from 2015

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    ...cognate peptide epitopes fused to the protein of interest are recognized and thus fluorescently labeled ...modified to use different antibiotic resistances, restriction enzymes and plasmid backbones. pUCXKT contains...fusion of the antibiotic cassette to the gene of interest. The gene to be expressed is amplified using a...gene-specific forward primer containing the desired restriction site (the MCS has several options) and a pUCX-specific...resistance cassette, a ribosome binding site, SacI restriction site and a linker region. The PCR product is ...used with other entry clones encoding the ORFs of interest: Buj et al., BMC Molecular Biology. 2013 August...A desired insert is cloned using traditional restriction enzyme cloning to create an Entry vector containing...

  13. 22 Hot Plasmid Technologies from 2014

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    ...method for protein inactivation Your protein of interest does x, but it also participates in y. Depletion...accomplished by knocking down your endogenous protein of interest and co-expressing its replacement recombinant ...into Golden Gate entry vectors via traditional restriction enzyme cloning, TA cloning, or annealing of oligonucleotides...would like to extend this work into their RTK of interest. Grusch, M. et al., EMBO J 2014 Aug 1;33(15)... genetic tag which can be fused to proteins of interest for EM imaging, or targeted to subcellular regions...sequence that effectively targets your gene/region of interest is a key step for CRISPR/Cas9 gene editing. Dr...users blog. New CRISPR plasmids available! Interested in purifying Cas9? Check out pET-28b-Cas9-His...

  14. Deep Mutational Scanning with One Pot Saturation Mutagenesis

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    ...pair of restriction enzymes. These enzymes, Nt.BbvCI and Nb.BbvCI, recognize the same restriction site, ...multiple amino acid changes affect a protein of interest’s function. This technique relies on the generation...deep mutational scanning.     3 reasons to be interested in deep mutational scanning Hundreds of thousands...variable bases (NNN) at any given codon site of interest can be cloned into an expression vector with Gibson...mutagenic primers that tile across a region of interest, generating multiple mutants in one PCR reaction...primer sets tile across the protein or region of interest. A low primer-to-template ratio is used to promote... be sense. Does the orientation of the BbcVI restrict site matter? Most of the time no, but there are...

  15. 5 Great Apps for Lab Life

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    ...information in the app is restricted to the NEB inventory only, the restriction enzyme sequence resource...smartphone nearby. We tailor these devices to our own interests with the content we store in them: music, emails...comes in handy when I use protocols that require restriction enzymes or PCR. The app contains a double digest...digest tool that is only helpful for NEB restriction enzymes since the instructions use NEB buffers. Nonetheless...name to discover all the information about that restriction enzyme or share it via email with a colleague...colleague. The app is also great if you’re adding a restriction site to a primer, you only need to skim the alphabet...screen to find the sequence for your enzyme of interest. Lastly, the Tm calculator helps you set up an...

  16. Plasmids 101: Screening Strategies Used in Plasmid Cloning

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    ...use restriction digest to screen your clones, but there are many other ways to choose restriction enzymes...Blue-white screens rely on vectors where the gene of interest is inserted into the middle of the lacZ gene, ...blue-white screen. Image from Stefan Walkowski.   Restriction digest Another way to distinguish between colonies...inserts from those that do not is by using a restriction digest. The key is to be strategic about your...on whether the plasmid contains your insert of interest or not. For example, choose enzymes that only ...route, check out our protocol video on using restriction digests to analyze plasmids, which can help you... you visualize this process, or visit our restriction analysis blog post. Colony PCR Colony PCR can...

  17. GCE4All: Making Genetic Code Expansion Accessible

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    ...If you study proteins, you’re probably quite interested in the canonical 20 amino acids. But in your ...your quest to learn more about your protein of interest, you may find the available amino acids a bit…limiting...Shandell et al., 2021).   How it works If you’re interested in using GCE, here are the basic requirements...plasmid will be needed to introduce the gene of interest containing the chosen codon that will result in...of the non-canonical amino acid at the site of interest (figure 1). While the codon used is often one ...getting high enough expression of your protein of interest. If that wasn’t enough, in this development-focused...introducing ncAAs with protein modifications of interest or any of a huge variety of ncAAs to study the...

  18. GPCRs: How Do They Work and How Do We Study Them?

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    ...TANGO) (Kroeze et al., 2015). PRESTO-TANGO uses a modified Tango beta-arrestin recruitment assay to measure... examples with plasmids deposited at Addgene.  PRESTO-TANGO: The ligands that activate many GPCRs are...Roth’s lab created an open source resource called PRESTO-TANGO (parallel receptorome expression and screening...this assay, binding of an agonist recruits a beta-arrestin-TEV protease fusion to a receptor-transcription...ligands for unknown GPCRs. You can find the entire PRESTO-TANGO kit at Addgene! TRUPATH: Bryan Roth’s lab... G proteins activated by ligand-bound GPCRs of interest. The TRUPATH plasmid kit consists of 20 plasmids...for more information on this system! GPCR-APEX: Interested in tracking the downstream signaling pathways...

  19. Advice on Career Paths and the Green Card Process for International Researchers and Entrepreneurs

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    ...to you as a researcher. National interest waiver The National Interest Waiver (NIW) allows for self-sponsorship...as the National Interest of the EB-1A. Furthermore, the ability to leverage the prestige of the employer...Cap Subject Industry H1B Academic H1B National Interest Waiver EB-1A EB-1B Cap subject industry H-1B...opportunities to develop skills related to their areas of interest. Additionally, since most universities and non-profit...determine if an applicant qualifies for the National Interest Waiver. The three prongs are: (1) The foreign ... and plan for future activities; including the interest of the greater scientific field, potential customers...must be able to show that you serve the national interest to a substantially greater degree than the majority...

  20. Visualizing Protein Turnover In Situ

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    ...-in of fluorescent tags into the gene locus of interest. An advantage of this alternative is the possibility...in situ. I was in luck when I attended a very interesting talk by Prof. Erin M Schuman, a former Caltech...antibodies directed towards two endogenous proteins of interests, suspected to interact, are added to a cellular... strand added to them. Only if the proteins of interest are in close proximity to each other can the DNA... one antibody was directed toward a protein of interest but the second antibody was directed toward AHA...antibodies recognized an endogenous protein of interest that also had incorporated the AHA tag after pulse...time. Thus, only newly synthesized proteins of interest were detected and later visualized by FUNCAT-PLA...
Showing: 161 - 180 of 755 results