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...idea. Our product team has implemented a policy of never rejecting an idea simply because it's not how things...brainstorm session is to start with something completely unrelated to the issue and consider how it could...lunch are just as valuable! In 2019, Addgene implemented a company-wide New Ideas Survey. Anyone could...generated 26 ideas, over half of which, when implemented, impacted the entire company. It is important...submitted the idea did not have to be in charge of implementing it. The purpose of this form was to encourage...head by giving yourself a quick, easy goal. For example, set a timer for five minutes and come up with ...’t have to be perfect or even good. The goal is simply to throw out ideas.  When your five minutes are...

...with ssDNA repair templates. Their work demonstrates that different repair template strategies activate...using dsDNA templates achieving ~1-10% efficiency. Easi-CRISPR uses a long ssDNA template to increase ...common lexicon for genome editing studies. These complementary goals are aimed at improving translation of ... loosely paraphrased in the tweet below. The complexity and challenges of homology directed repair (HDR... I think I may have finally grasped just how complicated DNA repair pathways are. I fully admit to being...working with CRISPR than just designing a repair template that contains your desired edit, and I want to...Editing in Human Cells Hands-on tips for repair template design from guest bloggers Dominik Paquet and ...

...abundance, consider the following example gel. Figure 5: An example gel (left) with bands corresponding...western blot requires a standard curve with a pure sample of your protein of interest (POI). This approach...need to know the exact quantity of your POI, but simply whether its concentration changes in response to...controls are equally strong, so the normalized value simply reports which POI band is stronger. The right gel...blot, and depending too much on normalization can amplify other errors in experimental design and gel quantification... a perfect world, protein concentration in your sample would directly correlate to protein abundance on...are highly expressed in cells. This means that at sample concentrations appropriate to detect a less-abundant...

...avidin can be added to your sample. Any free biotin-binding sites on this complex can bind to your biotinylated...of biotin-avidin complexes bound to your protein of interest leading to amplification of the reporter ... proteome that are biotinylated. In E.coli for example, the BPL, BirA, is responsible for biotinylating...discovered in the 1960s, strepavidin. The Biotin-avidin complex can then be used and/or manipulated for a scientist...immunoprecipitation experiments. Biotinylated proteins or complexes can be easily purified using columns or beads ...sites found on avidin are also ideal for signal amplification during these experiments. During detection, ...This strategy is referred to as the Avidin-Biotin Complex (ABC) immunohistochemistry (IHC). For more information...

...adopting simple note-taking tools, others using excel sheets or organizing folders in complex ways, there...lengths to keep accurate records, but given the complexities of modern research, it’s often not enough. A... have software tools that help us efficiently accomplish our daily goals. These tools remind us of our...if they are to switch from paper to digital for example): “… 99% of respondents indicated that ease of ...developed the SciNote Electronic Lab Notebook. Implementing an electronic lab notebook starts with defining...information. Does your lab need to meet any specific compliance requirements – e.g. 21 CFR part 11, HIPAA? Answering...crucial importance when applying for a patent for example, it also comes in handy if a supervisor wants to...

...desirable quality sought after by employers. These skills show employers that you can fully investigate ...are very desirable to employers because they save time and money and make employees more productive. Careers...investigate and solve a problem or complete a project, overcoming inevitable failures and setbacks. There are many...and formatting skills, which are useful to many employers. Increasingly, computer skills are highly desired...shows that you are passionate and capable. For example if the job involves traveling, showing that you...organised and experienced traveler by citing specific examples, may help you get your foot in the door. Match...buzz words’ to ensure recruitment agencies and employers put your resume forward for the role, because ...

...sense of the employee’s workload, updates on current projects, and offer feedback. The employee should ask...need to be decided. Figure out what you need to accomplish to get to the next stage, and then adjourn. ...communicate outcomes to the rest of their team. Here's a template for project meetings. Team Meetings: For standing...members still have their voices heard. Here's a template for team meetings. One-on-Ones: Managers should...should set up regular check-ins with their employees. 15-30 minutes weekly or monthly will usually suffice...you just need to give everyone a heads up (for example, notifying people of an updated protocol), send... positive peer pressure to make sure everyone completes their assigned items to keep the project moving...

... not robust. Sampling noise There is, of course, simple noise in the process of sampling and inference-making... to determine precisely how the methods were implemented. We run into this problem frequently over the...problems above: unclear methods, QRPs, and small samples, then when replications do not come out the same...statistical tools that generate a p-value.  As an example, if you hypothesize that growth rates will be higher...data and a new, preregistered experiment. Reduce sampling noise with registered reports, the gold standard... special emphasis on positive controls and good sample sizes, concerns that are too easy to overlook when... worked with citizen scientists to design and implement authentic scientific research and as the Director...

... desired mutation, then use those to PCR amplify the template plasmid. The parental, nonmutated DNA is...containing the desired mutations, to amplify both strands of a plasmid template. In addition to containing the...mutations, large deletions/insertions require complicated, often costly in vitro assembly methods. A new...nick repair. This method can work well, but poor template digestion can result in a high number of background...extension PCR is used to splice together two PCR amplicons based on sequence homology, and the subsequent...After being treated with DpnI to digest away the template plasmid, these PCR products are directly transformed...pitfall of REPLACR-mutagenesis is that you must PCR amplify the whole plasmid, which could introduce mutations...

... fragment of the NanoBiT® complex, which, upon binding to its complementary partner LgBiT, reconstitutes...disordered and structurally complex. The tumor suppressor TP53 (p53) is one example of these difficult-to-study...with nothing more than substrate addition and a simple add-mix-measure method. NanoBiT® luciferase activity...also be combined with the HiBiT tag to provide complementary validation of protein expression and localization...or co-cultured across very narrow filters, complementation in recipient cells proved that the enzyme was...and tissue processes, but their low abundance, complex regulation, and context-dependent activity can ...fluorescently-labeled VEGF-A to the heteromeric receptor complex. Find sHiBiT-KDR Fusion Vector at Addgene! HiBiT...

...plasmid monomer from a plasmid sample, or if no monomers exist in your sample, you could try linearizing ...control when performing a diagnostic digest is a simple but effective method for detecting plasmid multimers...Bedbrook & Ausubel, 1976). In fact, if your plasmid sample has a mix of different-sized multimers, you may...corresponding to each type of multimer present in your sample. In these instances, please note that the supercoiled...agarose gel). If the presence of a multimer in your sample remains unclear after visualizing it on a gel, ...perform whole plasmid sequencing of our plasmid samples during our quality control process. Since MiSeq...topologies and multimers present within a plasmid sample. Troubleshooting Tips   Figure 3. Summary...

...are having difficulty amplifying your target sequence, or if you want to amplify sequences from a large..., but if you are having difficulties in amplifying a template, particularly from a large genome, you can...Assembly, Golden Gate etc.), and try to find the simplest way to do it (i.e. avoid assembling too many fragments...Design primers that overlap at least 24 bp with the template and if the sequence has high GC or AT content,...Remember to quantify the DNA concentration of all the samples by gel or by nanodrop to assemble the  fragments...correct insert Sequence the regions that have been amplified by PCR, putting special attention in the areas...plasmid. Many vectors contain sequences that are complementary to standard sequencing primers. If not, design...

...reducing editing from ~30% in control samples to 0-10% in Acr samples. Acr proteins can also be used to prevent...assembly of the crRNA-Cas complex (Zhu et al., 2019). Inhibiting DNA cleavage Example: AcrIIC1 binds to the... of discovery (Bondy-Denomy et al., 2018). For example, AcrIIA4 mentioned above indicates that it’s the...activity one of three ways: Inhibit DNA binding Example:  AcrIIA4 blocks Cas9’s interaction with the PAM...site (Harrington et al., 2017). Inhibit crRNA loadingExample: AcrIIC2 interactions with Cas9 interferes ...and spatial control of CRISPR-Cas activity. For example, fusing AcrA4II to the photo-sensitive LOV2 domain...selectable markers in engineering viruses. For example, an Acr gene was used to replace genes in the difficult-to-engineer...

...“Vector Type Other” column. For example, you may enter “donor template vector”. Cloning Method Choose...plasmid deposit process can be broken down into four simple steps: Send plasmid information to Addgene Material... explain how to use our deposit spreadsheet to complete Step 1. Steps 2 and 3 are usually very easy — ...the Deposit Spreadsheet is the quickest option. Simply download the spreadsheet and copy and paste your...any functional consequences of that change. For example, “Mutated aspartate 123 to Lysine (D123K), lowers...contact Addgene. We may require that you send your samples to Addgene pre-transformed in this strain. ...and sending the deposit spreadsheet Figure 2: Example of the deposit spreadsheet showing the first eight...

...high and above what would be expected in patient samples (e.g.: blood, urine). Adapting toehold switches... dozen versions of the toehold switches and amplification primers. This helped make sure that we had a...up clear tasks for each member of the team to accomplish so that many elements of the project were able...involved definitely contributed to our ability to complete the project on such a short timescale. Our article...pathogen’s genome as unique barcodes to identify samples containing the virus. Of the 48 sensors, targeting...this includes primer directed isothermal RNA amplification, new toehold switch designs, CRISPR/Cas9-mediated... diagnostic results.  Our approach now detects samples at clinically relevant concentrations (1-3 fM; ...

...use this tool to detect a BRAF gene edit, simply PCR-amplify the BRAF locus from the edited cells (the...gRNA-transduced samples (data not shown). Pro-Tips: 1.Designing primers against genomic DNA to amplify the gRNA...and GFP-edited amplification products (BRAF-GFP duplex) and from GFP-edited amplification products (GFP-GFP... wanted to inactivate the gene BRAF (a kinase implicated in several human cancers) in A549 cells (a human...should contain the edited BRAF sequence), and PCR-amplify the BRAF locus from control cells (the PCR product...genome browser and Primer3 to design primers that amplified a 500-600 bp product. The primers I designed (...gel that highlights the important results (for example, if you needed to make a figure with the data)....

...essays in the journals Nature and Science, for example). This blog post is about how to get the most ...requires that all of the experiments have been completed. Therefore, to be ready to apply for a Group Leader... questions like: What figures are missing? For example, are additional controls necessary? As you build...Discussions can reveal mutual scientific interests, complementary perspectives, a shared communication style. ... week planning an experiment, even a relatively simple experiment. However, by taking this time, you can...master. While the calculus of these decisions is complex, here are a few guidelines to help you evaluate...result? Is there a tractable question or are you simply describing a phenomenon? Exploration can be important...

...homologous recombination. The concept is simple: deliver a template with a modified version of the targeted...homologous recombination using a repair template. These templates can include loxP sites to create conditional... sequence to the cell which will recombine the template with the endogenous gene. Here, we'll describe...homology is typical for targeting constructs. In the example shown in Figure 1, large sequences corresponding...is expressed (knocked in). As seen in the above example, you can remove a floxed resistance gene using ...plasmids described in the preceding sections are simple ways of knocking out a non-essential gene of interest...functions normally because one of its exons has simply been replaced with the same sequence flanked by...

...relatively easily synthesized, must be produced using complex mixtures derived from cells or using live cells...expression consists of transfecting cells with your DNA template of choice and allowing these cells to transcribe...systems Insect cells can also be used to produce complex eukaryotic proteins with the correct post-translational.... These systems enable high expression of very complex, glycosylated proteins that cannot be produced ...expression. Protein expression in bacteria is quite simple; DNA coding for your protein of interest is inserted...pET, pRSET, Gateway pDEST, and pBAD vectors for example. Protein expression from each of these vectors ... expression systems, including the majority of complex post-translational modifications. Extraction and...

...protein fusion. CRISPR genome editing can help accomplish this goal. The power of this system lies in the...individually generate gRNAs and homology arm repair templates containing the tag for each insertion. Also, it..., as well as the 5’ and 3’ homology arm repair template (Fig. 1, Step 1). There is no need for PCR or ...the second site accepts the homology arm repair template. SapTrap includes a prebuilt donor plasmid library...be used to generate functionally unique repair templates. A final ligation reaction correctly assembles...genetic locus, the toolkit also offers repair templates for tagging proteins in a tissue specific manner... CRISPR/Cas Plasmids - Protein Tagging. Beyond simply monitoring protein localization and activity, CRISPR...