...confidence that they aren’t simply doing an experiment wrong. However, even simple procedures like pouring...someone else's new and incredibly useful technique simply by following the abbreviated methods section found...research. I’ve talked to many researchers who have complaints about the unreliability of antibodies. IPI is...sifting through paper lab notebooks). Once you’ve completed your experiment, you’ll need to analyze your results...individuals and by organizations like Addgene (a good example is our series of blog posts on the defunct genome...promote reproducible publishing practices. One example is the publication of peer reviews so we can see... more likely that we’ll find a fluke when we’re simply trying to get our experiments to work, but part...

...binary systems. In recent years, a number of more complex systems have also been developed which don’t fit...that transcription is now OFF.   We’ll look at examples of both natural and engineered repressible promoters...transcriptional activation domain VP16 taken from the herpes simplex virus. tTA binds to promoters containing TetO elements...in mammalian cells, this system has also been implemented in a variety of other organisms, such as sphingomonads...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...systems above if they all behave very similarly?” The simple answer is that sometimes it is necessary or desirable...

...choosing antibodies   Complexity is a key factor Biological systems tend to be complex with a variety of ...than a broadly  reactive secondary (Fig. 1). For example, an anti-mouse IgG2a secondary would be a better... rabbit versus mouse monoclonal antibodies for example. Rabbit monoclonal antibodies have higher affinity...affinity binding, such as immunoprecipitations. Your sample species should also be considered when choosing...immunoglobulins naturally present in the tissue sample. There are species-on-species staining protocols...proteins interacting. Experiments tend to be equally complex with users trying to probe multiple targets in ... from each other. In a direct approach, that is simple - but what about when you need to use an indirect...

...membrane will remove some of your sample —and harsher stripping means more sample loss. If you are still optimizing... tool to identify a protein of interest in a complicated solution like cell lysate. But they can be a ...you want to detect more than one protein in your sample? Or what if something weird happened during your..., almost like new. This approach preserves your sample and saves you from having to run a whole new gel...stripping solution (see below) to minimize loss of your sample from the membrane. Next, plan out the order of ... the weaker binding ones first to preserve your sample. Pro tip! Because some protein is lost each time...antibodies on it if you plan to reprobe! Just as your sample protein is retained better, antibodies will stick...

...abnormalities due to the deletion (i.e. the plasmid complements the deletion). If the phenotype is corrected,..., well-studied and inexpensive. In addition, a complete set of well-annotated deletion strains is available... the absence of methionine (i.e. the plasmid complements the deletion). Find the Plasmids Here Plasmid...blotting in other experiments (Figure 2).   Complementation reveals conservation of function Students have...Addgene, to successfully analyze Met protein complementation. In the procedure diagrammed in Figure 3, students...promoter controls MET/Met gene expression, complementation should only occur when galactose replaces glucose...shown that most, but not all, S. pombe Met genes complement the corresponding S. cerevisiae met deficiencies...

...biotin-containing media. FRET is another example of a protein fragmentation complementation assay that can be used to...can be part of multiple dynamic and overlapping complexes that have distinct functions. Many existing methods...proteins that interact with a particular protein  complex as opposed to an individual protein. The Bethune... when the bait is part of a particular protein complex. Not performed in a native cell context. For AP-MS...experiment is called a protein fragmentation complementation assay (PCA). In the Split-BioID method, each...Protein Interactions. Split-BioID is a protein complementation assay where BirA*, an E. coli biotin ligase...proteins Y and Z interact, the two BirA* fragments complement each other to form a functional BirA* enzyme....

...with wild ones. For example, Drosophila lines with compound autosomes are completely infertile when mated...organisms with Cas9 risk creating a gene drive? The simplest way to make a gene drive is to insert an endonuclease...chromosome and its own gene is used as a repair template, thereby converting the heterozygote into a homozygote...But please be aware that there are many easy-to-implement confinement strategies that are robust to human...authorities) was to detail these robust and easy-to-implement confinement strategies2. Our hope was that anyone...laboratory organisms but not wild organisms. For example, an sgRNA-only drive will spread exclusively through...with that sequence2. Both methods are easy to implement and have been tested in yeast5. Ecological confinement...

... viral polymerase complex then recognizes the encapsidated minigenome as a template for replication and...functional viral polymerase complex and utilize the minigenome as a template. Thus, it is important to ...with the viral polymerase, which is formed by a complex of two proteins: the large (L) catalytic protein...-minigenome). The anti-minigenome is used as a template by the viral polymerase to synthesize additional...addition, the polymerase will use the minigenome as a template to transcribe the reporter gene (e.g. firefly ...studied independently of other viral proteins simplifying the interpretation of results. This system can...transfected into cells are also extremely important. For example, particular ratios of plasmids encoding NP and ...

...two hours were split up between several Addgene employees, who all made the daunting task feel more comfortable...to span the first three months of my six month employment. In the beginning, I spent a lot of time shadowing...everyone was very approachable.     Fig. 1: Examples of DNA sequencing QC work    In addition to...teams at the company. This provides new (and old) employees with information on how Addgene operates as a ...most meetings are scheduled to take place and employees are generally expected to be available. In addition... core hours) at a time that works for you. For example, if I needed to go to an appointment at 3 PM, I...finished on time, there were no issues. Another example of helpful flexibility was the ability to work ...

...safe to say the sample had their attention! They isolated DNA from the sample, amplified the CRISPR locus...lab often tests samples of new bacteria from anywhere they can find, whether the samples are from a rain...might be phylogenetically classified as the more complex Class I systems that contain multiple subunits,...Cas7-11 appeared to have derived from the more complex type III-A and III-B systems. Though it was composed...the domains into a single protein, shedding the complexity of its ancestors. They classified the system ... matter – the mutated copy could be replaced by simply dragging and dropping in a functional copy of the... also compatible with both AAV and adenovirus templates. The system, which is larger than Cas9, can also...

...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 ...

...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...

...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...

...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...

...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...