...that you want to use for your experiments and verify that your construct is functional. Advantages of the...more about the Gateway cloning method and its advantages. An introduction to Gateway technology The Gateway... clone and destination vector carry different antibiotic resistance markers (indicated here by plasmid...coding sequence. If you choose this strategy, it’s important to include the proper protein expression elements...Clone When making the expression clone, it is important to choose the destination vector that best fits...Gateway cloning kit, and MultiSite Gateway plasmids. Constant reading frame When you move a DNA fragment from...stays in frame. High efficiency The positive (antibiotic) and negative (CcdB) selection markers used for...

...inconclusive and unreliable. Proper controls provide the constant variables that enable the correct interpretation...of the independent variable you are testing. Importantly, they demonstrate the functionality of your experimental... the transfection reagents and process. It is important to note that optimal experimental conditions, ...Control (mentioned above) could also serve as an important negative control. In this context, the empty vector...interpretation of the results because it provides an important reference point when analyzing the specificity... same time. Although the replicate control is important, it speaks to the consistency of the reagents ...controls are more time consuming and ultimately more important. Ideally, each biological replicate should use...

...highest quantum yield donor, the highest absorbing acceptor and fluorophores with significant overlap ... theory, is an easy technique, but it is very important to follow some simple rules to avoid common pitfalls...purpose of your studies. These parameters are less important when designing a biosensor, as the distance between...and their spatial orientation will be fixed. The quantum yield of the donor (number of emitted photons per...extinction coefficient of the acceptor (linking the quantity of absorbed light, at a given wavelength, to the...Clover Green Mammalian Expresses Clover (a GFP variant) commonly used with mRuby2 pLSSmOrange-N1 Orange... mRuby2 Red Mammalian Expresses mRuby2 (a RFP variant) commonly used with Clover pGWF1 Cyan & Yellow...

... in March, he and his colleagues took another important step forward by dissecting that S. pyogenes protein...that SpyLigase can be used to link affibodies or antibodies against common tumor markers to subsequently ...cancerous cells expressing low levels of tumor antigen. I asked Howarth to tell us more about SpyLigase... to another peptide tag. Addgene: What is the advantage of peptide tags for building larger protein assemblies... shouldn’t get in the way much. Particularly we wanted to be able to build assemblies through covalent...stability really mattered for efficient capture. We wanted to be able to build a highly multivalent assembly...approaches to improve CTC capture, but I hope that our antibody tentacles may have an impact on this challenge...

...then collects the perfusion liquid for quantitation. The advantage of this method is that it can be performed...dopaminergic neurons in a brain region called the substantia nigra, which is the main source of the body’s...inserted into the region of the brain where DA quantitation is desired (Figure 1). The probe perfuses liquid...performed in a live awake animal by surgical implantation of the probe into a specific brain region. While... sensor. This sensor was engineered from a GFP variant and a DA receptor such that GFP fluoresces when...when the receptor is bound by DA. Two variants of the sensor, dLight1.1 and dLight1.2, exhibit high (submicromolar...GRABDA sensor based on a similar strategy: a GFP variant bound to a DA receptor, such that the GFP fluoresces...

...meeting participants. They were the ones holding poster tubes (or as our baffled flight attendant called...internal motto for scientists who contact us and want a guarantee that their experiments will work (yes, this...through TALENs and CRISPR-Cas nucleases, provided important context for the rest of the day. He was followed...curious about these newest features of our website.  I want to offer a huge thanks to all the Addgenies who ... helped get me to this meeting. And of course, I want to thank the organizers of this meeting who have...share their cutting edge science in increasingly important field.  Interested in Addgene's Keystone Poster...

...G418-selected clones. However, there are important disadvantages to the AAV-based approach that must be ...this post. Adeno-associated viruses (AAVs) make fantastic gene delivery vehicles for episomal gene expression...downstream of a functional promoter (gene traps). The resultant gene targeting system could be used to routinely...non-sequence specific frameshift mutations through aberrant double strand break repair, it has proven more...endogenous epitope tags, or the introduction of dominant mutations of a gene into the endogenous allele...nucleases In an effort to combine the respective advantages of the AAV and CRISPR-based approaches, several...generating human somatic cell gene knockouts using recombinant adeno‐associated viruses." Nucleic acids research...

...cells more resistant to chemotherapy. You can find these plasmids at Addgene. Another advantage of mice ...researchers: the mouse (Mus musculus). Mice have many advantages as a mammalian model organism for scientists ...nervous system. The fly has also emerged as an important human disease model with potential for therapeutic...in the fly (Pandey and Nichols, 2011). A huge advantage of using the fruit fly is the array of genetic...due to their simplicity, yeast have become an important experimental model in the study of neurological... 1960s, the zebrafish has gradually become an important model organism. They share about 70% of their ...Craig Mello a Nobel Prize in 2006. One of  major advantages of C. elegans is that they are transparent throughout...

...Broccoli, and useful for quantitative studies. Specifically, Corn was developed to quantify RNA transcription...that is generated allows for qualitative and quantitative measurement of processes including RNA movement...DFHBI can only be used for qualitative, but not quantitative, studies. Additionally, Spinach, the first aptamer... In the search for something more stable for quantitative imaging, Ming Hammond’s group created Spinach2...Gram-positive bacteria (Kellenberger et al., 2013). Other variants of Spinach include Baby Spinach, a miniaturized... flow cytometry, the Jaffrey group was able to quantify the effect of mTOR inhibition on the activity ...short non-coding RNAs (Autour et al., 2018). Three variants of Mango, coined Mango II, III, and IV, were then...

...for synaptic glutamate response. They generated variants optimized for different applications (v82 has ... and powerful high-throughput screen to select variants based on performance metrics specifically for ...vivo (Farahani et al. 2022). The authors take advantage of the SH2 domain of ZAP70 (ZtSH2) which binds...sensors Whatever your analyte of interest, it’s important to choose a biosensor that has a binding affinity...BeadScan platform for screening candidate biosensor variants for performance under two-photon fluorescence ...lifetime imaging. B) LiLac responses are highly resistant to pH changes. Image adapted from Koveal et al...tdTomato), they produce a temperature sensor that significantly outperforms previous designs. B-gTEMP is excitable...

...from their evolution. Southern blotting and quantitative real-time PCR Southern blotting, named for its...DNA identity, size, and abundance. If a scientist wants to know if an organism has a particular gene mutation...commonly used. In all cases, the result can be quantified by the strength of the signal in comparison to...experimental questions. Real-time PCR detects and quantifies a gene or DNA sequence of interest by recording...compare different DNA sequences to see which is more abundant in a sample. Whereas Southern blotting is labor...of high-quality DNA, real-time PCR has several advantages including easier automation, higher throughput... similar experimental questions (see below). Importantly, although their role has changed, restriction...

...optimized, it’s important to ascertain how long the rescue phenotype will last, and more importantly, if it does...genetic disease in a postnatal mouse model, an important proof of concept for future preclinical and clinical... time, which would prevent the accumulation of unwanted mutations over time. DMD is a good choice for ...does not cause obvious germline modification, an important finding given the controversy associated with ...great step forward for CRISPR gene therapy, it’s important to realize that DMD is a simpler case than other...and large animal models with mutations in human-relevant exons 45-55, rather than in the traditional exon...Potential off-target effects in muscle, as well as unwanted germline editing, must be rigorously examined ...

...impractical to catalog every variant of a feature. Instead, we identified common variants, and then crafted a ...principles. For every task, we envision what the user wants to do and make the path to accomplishing their goals...with every possible option, we place the most important controls front and center, and make specialized...sequences. These plasmids contain features such as antibiotic resistance markers and replication origins, but...threshold. This issue is addressed by adding more variants to the database. Another limitation is that by...augment the database with new fluorescent protein variants. The future of DNA feature identification What...to newly sequenced strains through our mismatch tolerant detection algorithm and thereby speed up the annotation...

...16528756. 2. Oh, Steve KW, et al. "Substantial overproduction of antibodies by applying osmotic pressure and...Rather than being released into the cell culture supernatant, the viral particles associate with the cell ...induce hypertonicity display enhanced monoclonal antibody production (Oyaas, 1989; Ozturk and Palsson, 1991...scientists rely on our AAV preparations for, we wanted to be certain that this supplementation strategy...test this approach with different capsids. Most importantly, because our AAV yields are improved we will ...18613082. 3. Oyaas, K., et al. "Hybridoma growth and antibody production under conditions of hyperosmotic stress...osmolarity on hybridoma growth, metabolism, and antibody production." Biotechnology and bioengineering ...

...going to tell you to pick up the pace Constant worry about grants and renewals What is different about...scientists start out in academia, it is especially important for graduate students and postdocs to get a perspective...interesting topics Having strong non-science skills is important for success Must always justify use of resources...outsource a task someone else who can do better, no grant writing Job changes can happen regularly (every ...learn about the diverse options and how to take advantage of them early is one way to smooth your path to...scientists following diverse career paths is an important first step. Developing mentoring relationships...enjoy the most. Having a better idea of where you want to go will help you focus on the transferable skills...

...background. In other cases, YFG or one of its mutant variants will need to be overexpressed or knocked down...disrupts the function of YFG. You could compare this mutant fly to one lacking the mutation in the same genetic...from having to create a new fly line every time you want to express YFG in a new tissue type (more details...For example, if you discovered five new genes and wanted to express them within a specific neuronal compartment...eg. LexA/LexAop and Cre/Lox systems) in case you want to express one gene in one cell type and another...process of generating your own fly lines. In the meantime, be sure to browse the publication references ... a means of altering cell fates and generating dominant phenotypes., Development 118, 401–15 (1993). PubMed...

...reaction. Table 1 compares some general advantages and disadvantages of luminescence versus fluorescence....studies utilized luciferase as a genetic reporter in plant and mammalian cells. Luciferase assays have since...Used In Concert with Fluorecent Proteins in Nano Lanterns Reporter gene assays A reporter gene, such as...bright than fluorescent proteins, but have the advantage of lower background levels and improved signal...expression. The regulatory element (wild-type or mutant) is cloned upstream of the luciferase gene in the... is then introduced into the desired mammalian, plant or bacterial cells. Expression of luciferase as ...and the activity measurement can then be used to quantify the effect of the regulatory element. Depending...

...a PhD student in his first year. “It was very important to me to be with them in the lab when they started...the lab and to take things slowly! It is more important to carefully select first hires who are well-suited... having projects ambitious enough that everyone wants to recruit rather than repel and hoard.” If you'... prepared me for what a PI without a personal assistant has to deal with. My inbox currently stands at.... “Being a PI is more than just experiments and grant writing. You have to enjoy the mentoring role.” ...every day and in the pub with us in the evening, wanting to know our latest results as soon as the gels ... build a strong, collaborative team. And take advantage of the many resources available from your institution...

...does one stand out amongst a pool of successful applicants and get chosen to work in his or her lab of choice...This is followed by some perhaps surprising, yet important, factors to consider when seeking your dream lab...the type of science we do fits in with what they want to do. The experiments must fit with their interests...short-term goals and chat about awesome science. Important factors in the search for a lab Studies show that...postdoc. Although the chosen research field is important, the end goal during this time is to be trained...role models and teach you to be the scientist you want to be. For much more on this topic see our blog ...neighbors during the interview process. What if I want a non-academic career path?  If you are considering...

...should I take to get to work?”; we often make important life decisions without much logic - even choosing... of work and communication before making this important decision. I’ll highlight some of these questions... if it is what is best for you. Some of these important questions will be easy to ask the lab head, and...the mentors you choose are teaching the stuff you want to know and display character traits and skills ...the department? Are they successful at getting grants and funding? Beware of bullying Your mental health...your aspiration? Will you help or at least observe grant writing? Get equipment quotes? Understand administrative... a career at a small, teaching college you might want to consider less expensive, portable areas of research...