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... you can download an annotated GenBank File or a SnapGene file for the sequences listed. You can then ...positions within the plasmid. The list of enzymes that can be detected is 5x greater than our previous mapping...cloning. With this more functional display, you can start thinking about your next cloning experiment...depositor provided a custom annotated GenBank file, this can be found on the main plasmid page under the "Resource...sequence For more immediate, high-level analysis, you can click directly on the “Analyze Sequence” button to..., but also have interactive functionalities. You can now hover over any feature, enzyme, primer, or ORF... product, and a description of its function. You can also click one location in the plasmid and then a...

...research practices (QRPs) can quickly lead us to find statistically significant results that are not robust...difficult to do?  There are four reasons that research can be difficult to replicate. Unclear methods Until ...false negatives in replications are unavoidable, but can be minimized with high powered research designs. ...population, new genetic variation, or different handling can all be important.  If we eliminate the problems above... out the same as an original research finding we can be more certain that some important, previously unknown...other materials. The use of reporting guidelines can also help remind us to include all of the essential...before conducting the experiment (note: registrations can be embargoed while the research is ongoing, but all...

...everything you do in the form of a protocol, and you can add in personalized steps like “check drawers for... next to each step as you complete it. Yes, this can be incredibly tedious, but it also works, so do it...organization of your workspace and your workflow can have a huge impact. Test different approaches, like... up your pipette tips to match your plate layout can help you track samples as you add them. Moving your...from your left side to your right as you use them can help you remember what's been added and what hasn't...hasn't. Developing simple habits like these can consistently reduce errors as you work, and the possibilities... If you need to focus, a large set of headphones can act as a Do Not Disturb sign, even if the headphones...

...occasional errors. That can be advantageous if you are trying to knock out a gene. One can also exploit homology-directed...people, we can use this kit to knock in those mutations. Once that's done in stem cells, we can differentiate...: For people unfamiliar with this new TALEN kit, can you describe it for us? Musunuru: TALENs are a type...type of genome editing tool with which one can introduce changes into the genomes of mammalian or other...is that the DNA-binding domain is modifiable and can be programmed to bind to essentially any DNA sequence...template for repair. By using these two scenarios, you can either knock out a gene or knock in a mutation. Addgene...more of an apples-to-oranges comparison. Addgene: Can you compare the TALEN kit to CRISPR/Cas9? When would...

...for viewing, editing or making plasmid maps, but can also analyze and annotate any DNA sequence. SnapGene... also has a suite of molecular biology tools and can make plasmid maps. Free for academic users. Serial...be “cleaned up” or changed in some way before you can analyze them. These tools help with that.  DNA sequence...Cloning ligation reaction calculators These links can help you calculate how much insert and backbone you...Calculator: This tool works for any cloning method. You can select from five different insert:backbone ratios...calculators Lab math calculators These calculators can help you with common calculations performed in the...iPhone Reagent and Data Repositories These links can help you find experimental and/or validation data...

...gene they control. Promoters can be organism or tissue type-specific, which can help you restrict the expression...palindromic nature, and high GC content. This instability can cause partial loss of an ITR during routine plasmid...recombination-deficient bacterial strains, such as Stbl3s. ITR loss can be detected by restriction digest using enzymes, ...companies now offer Sanger sequencing conditions which can read through this challenging sequence. Transgene...map, its sequence is located between the ITRs. It can be anything you like as long as it’s ~4.4 kb or smaller...transgene to a specific species or location. Promoters can also drive different levels of expression. For example.... Lox sites are directional 34 bp sequences that can flank the transgene and are located between the ITRs...

... aimed at a peer audience.  Note that lay people can and do consume scientific communication (it is, after...how one moves from layperson to expert) and peers can and do consume science communication.  Formal education... is provided, typically ad hoc, for learners who can choose to engage or disengage with a very high level... is field-specific terminology or language. This can refer both to terms that are only used in the field...non-jargon in the context of disease testing, it can still be considered jargon in most other uses. Many...that will amplify a sequence only if present.” You can see how you could end up in a very confusing situation...communications project without an audience?  An audience can be defined by any number of characteristics. Common...

...availability. An antibody can only bind to a target, or epitope, that it can access. If the epitope is...antibodies, which only recognize one epitope. (It can sometimes be an issue with polyclonal antibodies,...used as a marker for flow cytometry, for example, can only be recognized by antibodies that bind to the...a cell membrane.  Picking the Right Antibody  It can get even more complex if you’re looking for proteins...to translocate or fold in certain conditions? It can help to visualize an abstract protein during your...change depending on the conditions and the assay can help you avoid a great deal of frustration when designing...designing and troubleshooting immunoimaging assays. It can also elucidate information about what a given protein...

...experiments, just about anything can disrupt your bench work and sometimes no culprit can be found…. In our lab ...experiments in Brazil can have the same issues as one doing experiments in France. We can learn with each other... knows that there are many ways your experiments can go wrong. Whether it be a bad fridge freezing your...discovered. As we started building, we thought – how can we make the connection between LEGOs and science ...differently than we do day to day. We discovered that it can be good for you and your experiments to think outside...Additional Resources on the Addgene Blog Learn How You Can Use Video to Share Your Research 5 Reasons to Use...

...publishing posts can be addicting. Even more satisfying, because Addgene is a non-profit, we can focus more ...but instead can involve walking alongside it and giving it a high five. While screaming can be therapeutic...visit scientists in person to figure out ways we can make Addgene better and make scientists’ lives easier...many young scientists are led to believe that they can all end up in academic positions (I’ve even blogged...plasmid deposits, and trying to learn ways Addgene can improve in its mission to make research easier. I...outskirts, and entirely outside of academia. These roles can include positions, like mine, that help support academia...

...they need to do before they can be good at it. Like any other skill, it can be developed with education...convert to the principle that you can't motivate people to do things, you can only demotivate them. The primary...if you are good at science and you are smart, you can be a good manager. The kind of smarts and the type...above – what type of bad manager have you had?). You can avoid some of these common pitfalls of management... an extreme by scientists) Are highly mobile and can move to a new workplace if opportunities for learning...primary obstacle of job dissatisfaction, the work can get done, and get done well. The rest of the blogs...series will address more specific tactics managers can utilize to be in touch with the needs of the team...

...screening project can be a tremendous undertaking, producing a wall of results that can only be described...kinome is a well-annotated collection of genes that can provide a useful starting point for developing a ...two sets, each with 4 gRNAs per kinase, so screens can be performed with either 4 or 8 gRNAs per gene. Further...more gRNAs found in genome-wide libraries, and thus can be screened in pooled format at a more-typical scale...plasmid formats A first for a library at Addgene, you can also order the individual lentiGuide gRNA plasmids...individual plasmids separately. Additionally, this set can be used for cherry-picking individual gRNAs that ...

...method that can handle all applications of iPSCs, but there should be at least one method that can help you...transgene-free and vector-free cells No genomic integration Can be difficult to fully remove virus from cells Plasmid... many variables (cell type, criteria used, etc.) can effect reprogramming rates and such a number maybe...also creates heterogeneous iPSC cell lines, which can complicate comparisons made between lines. Incomplete...polycistronic vector or multiple single-gene vectors can be used to package reprogramming factors. An important...re-expression of viral transgenes in iPSC-derived cells can interfere with their differentiation potential. Finally... as well repressed following reprogramming. This can be overcome by using a Tet-inducible virus to allow...

...the potential CRISPR technology can have on the human microbiome. You can consider the microbiome as a collection...selection step for successful modifications, efficiency can be low, especially for larger modifications. What.... The system also functions in S. pneumoniae and can be used to generate multiple mutations simultaneously...takes two days, and the pTargetF and pCas plasmids can be subsequently cured from the bacteria. Although...using BbsI and Golden Gate Assembly. Either plasmid can be also linearized with XbaI to insert extra elements... a multi-spacer CRISPR array, CRISPR-transposons can be multiplexed. Transcriptional repression (CRISPRi...dead (dCas9) targeted to a promoter or gene body can repress transcription by physically blocking the ...

...color emission. As you can see in Figure 1, emission spectra of neighboring colors can and do overlap. For...and the markers they label) can occur. As I mentioned previously, you can also use fluorescent reporters... inside of your cells. While proteins themselves can emit intrinsic fluorescence when excited with ultraviolet...only occur in combination with that reporter, you can infer its presence through detecting the reporter's...combinations for different markers of interest, you can separately detect these markers in your analysis.... photons excite electrons of a fluorophore, they can do so at a range of wavelengths, rather than at just...names, for example, BV421 and RB545. Fluorophores can also come in combination of two conjugated single...

...expressed genes can be the next stage in making sense of such data. These targeted screens can allow a researcher...tools to study the immune system and cancer. More information can be found through https://wermelinglab.com...Medicine, Solna, Karolinska Institutet, in Sweden. It can be very time consuming to design 5 guide RNAs (gRNAs...CRISPR screen. Luckily, the Green Listed software can help you do just this, probably in less than a minute...properties, migration abilities, or other phenotypes that can be efficiently measured at the single cell level)...receptors expressed on the cell surface. Thus, a screen can be used to target surface proteins to determine which.... Once you have your list of genes, Green Listed can help make the gRNA design process quick and easy....

...the candidate off-target sites. For example, if there are 10 putative off-target locations you can 1) ...sequencing methods. Candidate site sequencing Sites with high sequence similarity to the gRNA can be sequenced...sequenced. These sites can be selectively sequenced to determine if there are significant editing events, serving.... In this blog, we will discuss when off-targets can be an issue in CRISPR, how to predict and minimize... nucleases are promiscuous. In other words, they can sometimes localize to unintended sites with sequence...reduce off-targets and patient risk. Hopefully you can understand that depending on your platform, off-target...predicted frequency of off-target events, many of which can be combined to lower them even further. While not...

...The localization of the fusion protein can be verified and it can be compared to what is expected based...catalytic activity or interactions) can be challenging. If a fusion can complement a knock-down or knock-...the chimera are encoded by DNA. Since researchers can generate almost any DNA sequence in the way that ...the native properties of the protein of interest can be challenging. In this blog I discuss strategies...derived from Aequorea victoria GFP sticks out and can be considered a linker (figure 1). In fact, for several... and N-terminus of the donor fluorescent protein can be truncated to increase the FRET efficiency. Creating...fusion,” but I find this very confusing because it can mean that the fluorescent protein is at the C-terminus...

...thereby generating a complete, circular plasmid that can be easily maintained in a variety of biological systems...proper orientation downstream of the promoter, you can add an EcoRI site just 5’ of the start of the gene...site just 3’ of the end of the gene. This way you can then cut the plasmid backbone as well as the insert...experiments.     Alternatively, this whole process can be completed using a single enzyme if your insert... that enzyme’s restriction sites, but the insert can then anneal to the backbone in either a forward or...Many companies now sell fast digest enzymes that can digest large amounts of DNA in as little as 10 minutes...migrate slower than smaller linearized fragments. You can separate your backbone away from any inserts cut ...

...high accuracy. Conversely, you can only run one sample at a time, which can become costly and impractical...is generally cheaper if you have many samples and can output more data, and it typically results in high...structures, or repetitive regions. Short-read sequencing can also be time-consuming, depending on the library ... to hundreds of base pairs, long-read sequencing can read sequence lengths in the thousands or even millions...by the instrument changes. Base-calling software can then identify the nucleotide that passed through ...technology. Created with BioRender.com. Nanopore can provide longer read lengths of 10 kb to 100 kb (or... to sequence an entire plasmid in one read. Runs can be completed in anywhere from under an hour to 72...