...colonies, check that the antibiotic in the plate correspond to the antibiotic resistance marker present...hopefully obtain your coveted plasmid with no substantial delays. Design, design, design Before diving ...fragments. You can generate the parts of DNA that you want to assemble together in a combination of different...difficulty amplifying your target sequence, or if you want to amplify sequences from a large genome organisms...successful and ultimately faster cloning. Remember to quantify the DNA concentration of all the samples by gel... experimental design. Full lawn of cells. The antibiotic in your plate might not work. Check the plates...E. coli  strain known to be sensitive to that antibiotic, and if the strain is growing fine, it is probably...

...are added to a cellular sample. Both antibodies have a short antibody specific DNA strand added to them....PLA where one antibody was directed toward a protein of interest but the second antibody was directed ...and, because I was studying a synaptic protein, I wanted to measure it’s levels in its normal neuronal context...designated work areas and come with health risks. Importantly, with this method it is not possible to follow...most common approach. With a good and specific antibody and the proper microscope setup we can observe...fluorescent tags into the gene locus of interest. An advantage of this alternative is the possibility of real-time...interactions in situ. In the standard PLA assay, primary antibodies directed towards two endogenous proteins of interests...

...between studies is substantial (e.g, the poor performance of mRuby2 is detected by Constantini et al (2015) ...steadily increasing. Of course, you want the ‘latest and greatest’ variants with the best properties for your...dimerization tendency, as is present in EGFP. Constantini et al, (2012) developed a cell-based assay that...If homodimerization occurs (most likely in an antiparallel configuration), typical structures known as ...OSER) whorls are visible. These whorls can be quantified for individual cells, and the number of 'normal...tagRFP does not act as a true monomer in cells (Constantini, 2012), whereas it was previously concluded to..., good performance in the OSER assay is not a guarantee that using this fluorescent protein to tag your...

...only the most important results to your audience. With a poster, you have the advantage of being able ... sections as a primary research article, it’s important to understand that presenting your work in poster...certain posters resonate with you (or not). It is important to reflect on these impressions when thinking ...present at your poster at all times, so other participants can identify you. You may also wish to make ...particular styles recognized to work well. It is important that your readers can navigate your poster and...and acknowledgements sections are inserted. The advantage of this style is that it will display your data...pattern across the poster. This style has the advantage of being different and potentially more catchy...

...sequence are important considerations for designing your gRNAs. For indels, it's not so important what location...nom. Me love gRNAs! Comic by Maya Kostman.   Important considerations before you start an experiment ...tool among them. While this seems obvious, it is important to remember that the same is true when designing...location in the gene you target, but it is important that your gRNA sequence is designed to be highly active...from). Finally, for HDR, location is much more important because you have to target within ~30 nt of your...to the intended edit rather than some passenger variant that came along with the single cell clone (although...SaCas9, NmeCas9, Cas12a enzymes, and engineered variants thereof offers additional PAM options that can...

...cloning a gene into an expression vector, you would want the start of the gene to end up just downstream ...verify that the insert ended up in the direction you want - usually by Sanger sequencing or further restriction...some DNA during the gel purification step, it is important to digest plenty of starting material. We recommend...be cut with both enzymes, and therefore it is important that the digest go until completion. The time ...running a gel for purification purposes it is important to have nice crisp bands and to have space to ... your favorite gel purification method, it is important to determine the concentration of recovered DNA...DNA in a standard ligation reaction. You ideally want a “recipient plasmid : insert ratio” of approximately...

...great improvements, not necessarily quantifiable, but always significant. Doubts, fears, insecurities… all...professionals has made possible a significant qualitative and quantitative expansion in the 2015-2016 academic...how to write cover letters, and has suggested important things to emphasize in order to be selected in...to Spain at one point, since I am not sure that I want to pursue the academic track.” How does an International...Scholars need strong proactive mentors who have relevant expertise and skills, positive disposition and... of Oviedo, the European University Miguel de Cervantes, the University of Navarra, and the University...pursue them, increased their network of contacts important for their career goals, and identified possible...

... and meaningful way it is important to choose the appropriate quantification methods and tools. It is ...living cells and whole organisms. You can take advantage of the many Fluorescent Proteins (FP) available...Once you've created your fusion protein, it's important to validate its function relative to the untagged...protein visualized with a fluorescently-tagged antibody. By employing “marker proteins” which are known... of a cell. This mapping can be done by using antibodies targeted to the marker proteins, however, in ... cell membrane permeabilized in order for the antibodies to reach their intracellular targets. Therefore... pipelines. Image analysis software produces quantifiable results and statistics that can be used to compare...

...therapeutic purposes. Both types have their advantages and disadvantages. Here is a table listing some of them... system which combined the advantages of both while limiting disadvantages led to the creation of induced...essence, pluripotent cells can be thought of as descendants of totipotent cells. An example of pluripotent... mostly from abnormal and/or supernumerary preimplantation embryos that are produced during in vitro fertilization...these multipotent stem cells are located in small quantities in specific “stem cell niches” found in many ...during ESC isolation. Low Chances of post-transplantation rejection when used therapeutically High Low...similar functional utility, iPSCs bypass the transplantation compatibility issues and ethical concerns of...

...Medicine. It’s not always easy to figure out what you want to do after graduate school, at least not while ... into my PhD in Molecular Biology, I realized I wanted a career in science outreach: engaging the public...mentor and graduate office. I cannot stress how important they were to my success as a student. They didn...supportive of your career goals, find people who are. I wanted to do fun science activities with the public, so....  Try new things to further your career Take advantage of opportunities provided by your programs and...including workshops and networking events. I took advantage of as many of these resources as I could. Networking...front work was done for me. I just had to take advantage of it. Blog and tweet I started my blog, Born ...

...gene systems, what do you want to make? So, tell us readers, what do you want to make?   References: Jerome...were so much fun and so incredibly useful that we wanted to make them free to use by everybody. (For more...YouTube primer on BIL gates.) Addgene: Why is it so important to you that these parts be made freely available...out of 15 are free. Now, the BioBricks Foundation wants to develop a language for programming life. We can...information into actionable signals. Let's say I wanted to detect every primary metabolite in a cell. The...language is put into practice? Endy: I think it's important to understand that these platforms, or pieces ...incredible is the bioeconomy is already pretty substantial. It's estimated that genetic engineering as applied...

...sense, genome replicative intermediate (anti-minigenome). The anti-minigenome is used as a template by the...effectiveness of antiviral compounds which often target the viral polymerase. Technical tips It is important that ...viral proteins are provided in trans (see Other Important Components of EBOV Minigenome System). However...negative sense RNA viruses (reviewed in 2, 3). Other important components of the Zaire ebola virus minigenome... Firefly luciferase can readily be measured to quantify (indirectly) transcription performed by the viral...utilize the minigenome as a template. Thus, it is important to choose cell lines that are highly transfectable...plasmid transfected into cells are also extremely important. For example, particular ratios of plasmids encoding...

... single domain antibody fragments typically engineered from heavy-chain only antibodies found in camelids...camelids. They possess many advantages over conventional antibodies: they are small (12-15 kDa), stable, ..., allowing for quantitative analysis using different detection methods. Since the anti-GFP nanobody is... RNA templates to DNA but suffer from several important shortcomings including low processivity (ie. the...MarathonRT, isolated from Eubacterium rectale, significantly outperforms other RTs in this regard and is ...still just as specific to their target as regular antibodies. These properties make nanobodies easier to transform...toolkit includes a set of plasmids each containing an anti-GFP nanobody fused with a different functional element...

...becoming more reliant on technology, and good computer skills will give you an advantage. Strong writing...leaving academia, but don’t quite know what you do want to do or even what careers are available to scientists...your goal might not lead to a career you actually want, you can feel lost. Use this post to explore the...including: Livestock Breeder Scientific Illustrator Plant Breeder Zoological Worker Conservationist Documentary... Statistician Bioinformatician Science Writer Grant Writer Journal Editor Science Marketer Software... who currently in the position. Questions you’ll want to find answers to include: Will you be sitting...sitting at a desk all day or be on your feet constantly in a lab? Will regular travel be required or will ...

... is possible that you may not want the plasmid depleted, but still want to use transient transfection ...Epstein–Barr virus (EBV) nuclear antigen 1 (EBNA1) or the SV40 large-T antigen (293E or 293T cells), allow ...mammalian cells is fairly straightforward and the resultant cells can either express the plasmid DNA transiently...reduces the rate of plasmid dilution but does not guarantee 100% transfection efficiency. Stable transfection...plasmid contains a cassette that will make cells resistant to a toxin), whereas negative selection would ...Before doing a transfection experiment, it is important to determine the proper concentration required...reagent). Cells should die within 3-5 days and resistant colonies appear in about 10-14 days depending ...

...PMID: 18193049.  9. Filipe-Santos, Orchidée, et al. "A dynamic map of antigen recognition by CD4 T cells...with the engineering of fluorescently labelled antibodies made it possible to track cells from one organ...harvesting lymph nodes at different times after antigen challenge, scientists were able to track where ...diverse tissue environments. These studies provide quantitative measurements of cellular motility, interactions...By specifically labeling T cells, B cells, and antigen presenting cells, scientists have been able to ...the bloodstream to invade lymph nodes and scan antigen presenting cells, but two-photon microscopy enabled...scanning was and how fast T cells could jump from one antigen presenting cell to another. Indeed, we now know...

...if you cannot convince others that it is important and relevant to them.   When you think about it, virtually...this lead to the conclusions I want them to draw and the perspective I want them to form? Training in science...believe that communication is the single most important skill that scientists need to succeed in their...next interview or whether your receive the next grant you apply for. After all, the only value your work...high level to their team.  When you're writing a grant, this means convincing a committee that your research...your audience about the work you've done. If you want to maximize the impact of your communication, you...you will also have to explain why the work is important and how it should change how the members of your...

...the most recent advances and how some of these variants may be appropriate for your specific research ...nuclease for your experiments, there are three important factors to consider: site accessibility, specificity...potential for off-target cleavage should be an important consideration during any experiment. Sensitivity...isolated from different organisms and engineered Cas9 variants that were able to target different PAM sites (... be targeted (Hu et al., 2018).  These altered variants can allow for targeting of your genomic loci of...stringent PAM specificity. This can be especially important for base editing techniques, where Cas9 must be...many options! Comic by Maya Kostman. Another important consideration when choosing a Cas9 is balancing...

...In this blog post, I will go over some advantages, disadvantages, and examples of how scientists are using...commitment. Another advantage is that this process makes it easy to generate wild type and mutant constructs at... common, and for good reason. They offer many advantages over the traditional restriction enzyme cloning...example of using Gibson assembly, imagine that you want to insert your gene of interest into a vector with... have the tag already included in the vector you want to use. If you inserted these two pieces of DNA ...such as a hairpin or a stem loop (as might be anticipated to occur within a terminator sequence), since...specific for each fragment as well the fragment you want next to it, so this doesn’t allow for mix and match...

...mammalian cells: easy to use biological tools are abundant! In this post, we will review tried-and-true protein...well as explore some new innovations. Whether you want to turn things on or off, permanently or temporarily...requirements To pick the best tool for the job, it’s important to know what the job requirements are. Here are...temporary control (a few days) be enough, or do you want a permanent control mechanism? Where is the control...together. Turn it off Turn it off forever If you want to turn off protein expression, you are almost certainly...for genomic delivery and have many therapeutic advantages, such as tissue-specific targeting. Looking to...1 CRISPR activator and repressor mechanisms Want to transiently induce expression? Check out Addgene's...