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.... After ligation, the insert DNA is physically attached to the backbone and the complete plasmid can be... are what allow the vector and insert to bind to each other. When the sticky ends are compatible, meaning...on either side of the vector are compatible with each other, the vector is much more likely to ligate ... the ligation will vary depending on the size of each and their concentration. However, for most standard...you want, and it will tell you exactly how much of each to use. Reference Page Top Index...

...copies of VP64 at each loci of interest, this allows more transcriptional machinery to be recruited per...TALE proteins, proteins had to be re-engineered for each gene, making wide-scale gene activation seem next...activation: rather than requiring protein engineering for each loci, CRISPR/Cas systems only require changing the...Guided by dCas9, VP64 recruits transcriptional machinery to specific sequences, causing targeted gene regulation...levels. Although other methods have been able to achieve much higher activation, dCas9-VP64 is great for...Description Rather than using a single copy of VP64 per each dCas9, SunTag uses a repeating peptide array to ...JL, Davidsohn N, Housden BE, Perrimon N, Weiss R, Aach J, Collins JJ, Church GM (2015) Highly efficient...

...that you come into the lab wishing there was a machine that could make your life a hundred times easier...off our hands. Good news! There is probably a machine or robot out there that does exactly what you want...lot of time. Enter the Biomek FXP! We wanted a machine that would improve our transformation protocol ...played a factor in our decision to go with this machine, but we also really liked that Beckman was flexible...apart before plating. With this fine piece of machinery we have been able to transform 96 samples in about...samples at the same time as opposed to preparing each sample individually. Enter the BIOROBOT! This robot...lab team. Enter the Liquid Handler!     This machine fits on a table top and can be easily programmed...

... the fluorescence amplitude measured for each droplet in each well (Figure 2). A clean ddPCR should have...a fresh one for each set up.  Wipe down the bench and all consumables with 10% bleach.  Pipette slowly...20,000 droplets using water-oil emulsion technology. Each droplet contains the ingredients for amplification...dilutions onto our ddPCR plate. As the titer is unknown, each dilution should fall within the range of the assay...emulsion containing approximately 20,000 droplets. Each droplet contains the material required for a mini...plate and measures the fluorescence amplitude of each one. Droplets that fluoresce contain the amplified...is common. Here are some steps you can take to achieve a clean NTC: Have a dedicated bench with a dedicated...

...Ig) heavy chains and two Ig light chains make up each antibody molecule. The Fc region and part of the...IgG, or IgE). In the context of the immune system, each isotype is expressed at different times of the immune...system production and many more engineered methods. Each type of antibody and production strategy has advantages...advantages and disadvantages compared to each other. Polyclonal antibodies These antibodies are derived...recognize multiple epitopes on the protein. However, each animal will make antibodies against different protein...full antibodies would not physically be able to reach. While a full antibody is ~150 kDa, a nanobody is...similar concentration of cell lysate was added to each well. Image from Sun et al., 2010.   Enzyme-linked...

...presence of that metabolite (Fig.2). Spinach eventually evolved into Spinach2, a more stable and brighter version...leading to a positive signal. The same effect can be achieved for a fluorophore that relies on dimerization ...lack of fluorescence for readout may render this approach less sensitive in most applications. Learn About...cleavage and an RNA aptamer that bound the compound malachite green (MG) to create a non-protein based visualization...essentially mimicking GFP.  Though the aptamer, called Spinach, required longer exposure time than its protein...possibilities. For instance, further selection of Spinach with a target metabolite creates an aptamer that...also has a higher melting temperature than its Spinach ancestors, and has a lower magnesium dependence...

...adeno-associated virus (AAV), from reaching their targets. The AAV9 serotype can breach the BBB following injection...but requires a high vector dose and is unable to reach all regions of the CNS. PHP.B, a neurotropic variant...C57BL/6J mice following IV injection but fails to reach the CNS of BALB/cJ mice. These results were surprising...C57BL/6J mice strains to identify variants that reached the CNS following IV injection. Compared to AAV9...antigen-1 (SCA-1), is the receptor used by PHP.B to reach the CNS; yet LY6A is an unlikely candidate. It was...transduced by PHP.B. The Deverman Lab took a broad approach and searched for genetic difference between the...phenotype in mice. They took a classic genetics approach and interbred C57BL/6J and BALB/cJ mice to map...

... express a gene of interest. While the general approach for using lentivirus, infect and select, seems...mycoplasma can be extremely hard to detect with levels reaching 108 cells per ml before the media becomes cloudy... cell lines, ELISA, fluorometry, or PCR-based approaches to detect even low levels of contamination. For...Uphoff et. al. outline a do it yourself PCR-based approach complete with freely available positive controls...benefit of the multiple harvest strategy is that each day you are supplying your producer cells with fresh...envelope. One drawback of the multiple harvest approach is that it tends to result in a higher volume ... your lentiviral prep!) and infect. While this approach is a great start to a successful infection, one...

...image and the resolution your microscope can achieve. You achieve the highest resolution when your image is...the image is further magnified before it finally reaches your eyes. These lenses determine the magnification...wondering why there are multiple objective lenses. Each objective performs essentially the same task but...observations. Some microscopes have digital cameras attached to them that allow you to capture images directly...

... was weird like that,” says Caitlin. A love of teaching also runs through Caitlin’s blood. “I come from...faculty members, I helped grad students who were teaching for the first time. I walk[ed them] through their...them advice, [and helped] proctor exams.” These teaching experiences led Caitlin back to being a student... signed up for a 10 week class at Iowa called “Teaching Your Research.” This course has participants take...you know about it [your area of research], and teaches you how to design a course, how to design good ...ground up.”  Science writing and editing These teaching experiences definitely weighed in Caitlin’s post-grad...but in the end, writing won out. “I considered teaching at a liberal arts school but I’ve always enjoyed...

...sample of your protein of interest (POI). This approach is reliable, but it’s overkill in many cases. ...normalized to the loading control: the intensity of each POI band is divided by the intensity of the loading...Figure 2: Normalization with loading controls. Under each lane, the fraction of the POI band over the loading...detected with an antibodies, just like the POI. This approach is so common that we’ve used it for our cartoon..., 2008; Gilda & Gomes, 2013). Depending on the approach you choose, your loading control measurement will...on the same gel or membrane (depending on your approach) as your final blot. Quantifying the entire lane...targets. Convenient! Comparison of loading control approaches Cartoons are nice and all, but we’ll leave you...

...that many of my old suitemates stopped talking to each other if I was not there to arbitrate for them. ...sell them. Types of Conflict Before explaining my approach to conflict resolution, it’s important to recognize...occurs when two people cannot agree or get along with each other, regardless of the situation. If left unchecked... of conflict can lead to people disagreeing with each other just for the sake of disagreeing. It can be...people or teams who normally get along well with each other disagree about a specific decision, direction...responsibilities. Knowing which aspects of customer support each team “owns” is critical for day-to-day operations...everyone walk away from a conflict happy, feeling they each got enough of what they wanted. However, a poorly...

...immunofluorescence There are a couple different approaches to be familiar with when talking about IF, direct...antibody targets the antigen of interest but is not attached to a fluorescent molecule, instead a secondary...BioRender.com   Indirect IF is a much more common approach than direct IF, so we will focus on this technique...why generating fluorophore-conjugated versions for each primary antibody could get out of hand. Instead,...can also require some additional optimization as each antibody performs differently and some antibodies.... There are multiple methods for fixing samples, each with its own pros and cons. The method you use will...immunofluorescence procedure with key considerations about each step. Image created with BioRender.com   The...

... was delivered to each cell, future applications could deliver multiple TFs to each cell, allowing for...The Zhang lab generated a comprehensive library of each unique TF open reading frame (ORF), barcoded for...target cells as a pooled library, which will amplify each TF to determine how its presence influences cell...atlas of directed differentiation To understand how each human TF can contribute to differentiation, the ...embryonic stem cells with the viral TF library, with each cell receiving only 1 TF expression construct. Cells...screening mechanisms, although the unique barcodes for each TF help streamline identification. While the Zhang...and/or TF dysregulation. A targeted TF screening approach can not only lead to a better understanding of...

...trafficking and their detection can suffer from photobleaching and cellular autofluorescence (Tsien, 1998)... typically occurring with a fluorescence-based approach. These challenges can include background autofluorescence...plates or media, which can obscure weak signals; photobleaching from the excitation light, causing the signal...look at several applications of this technology. In each section below, we cite the original paper but link...context-dependent activity can render traditional tagging approaches insufficient. Vascular endothelial growth factor...) and by endocytic trafficking of the receptor. Peach et al. (2021) utilized HiBiT to address these challenges...kinetics of the VEGF-A and the VEGFR2-NRP1 heteromer (Peach et al., 2021). They used HiBiT and LgBiT fusion ...

...Capturing), if you ask me.  Each of the three steps need to be optimized for each specific sample type. And...including epigenetic modifications. But it's not an approach for the faint of heart!  The Process ChIP can... your protein:DNA complexes will remain bound to each other until you are ready to separate them. Most...Most proteins are not likely to remain attached to the DNA throughout the sample processing and therefore...sample may need more or less cross-linking to firmly attach the proteins to the DNA, depending on how tightly... idea to add "patience and an extra-methodical approach" to the top of your protocols when embarking on... Rachel Leeson...

... from each other, and a lower percentage gel will help separate larger bands. 10% Rule For each sample...and Running an Agarose Gel: Add loading buffer to each of your DNA samples and mix well. Use 5 µl of loading...run the gel without EtBr in the buffer you will reach a point where the DNA will be in the bottom portion...manufacturer's instruction will tell you the size of each band), you can infer the size of the DNA in your...

...sequences (loxp and lox2272) and can not recombine with each other. When Cre is introduced to the system it binds...optimal/minimal injection titer and volume needed to achieve adequate expression in your cells of interest.  ...injection titers and volumes based on the serotype Since each serotype also exhibits distinct patterns of spread...waiting periods and/or higher injection titers to achieve adequate expression. Conducting a time course experiment...start an experiment before adequate expression is reached you may obtain a false negative or conclude the...to minimize off-target expression while still achieving adequate expression in your cells of interest..../or injection volumes may need to be tested with each new vector or lot used.   Following this stepwise...

...knockin or knockout mice. While these techniques are each useful for introducing one modification into the...engineering should be used to separately introduce each mutation independently into different mice, and ...have a combination of the mutations engineered into each of their parents. Here’s how to cross mice, using... scheme should start with parental mice that are each homozygous for one of the two mutations that you...Genotyping involves getting a small piece of tissue from each mouse (typically via either a blood draw, tail clip...in handy here! Crossing your F1 heterozygotes to each other will generate approximately 1 in 16 mice that...gametes that contain a mix of genetic material from each of the parents. If both of the mutations that you...

...organizers and moderator should have a well-planned approach to making the event interesting and engaging. ...an organizer) should set up a 30 minute call with each panelist to get to know their style and to get their...discovering interesting stories, riffs or opinions that each panelist can contribute. The moderator should identify...information and remind them about the event as it approaches. Room set-up and logistics To make a really...formality and distance that prevents interaction with each other and the audience. Put the panel on a stage...panelists in a slight semi-circle (so they can see each other) on comfortable chairs (stools if you have... better. Have a good microphone system ready for each panelist if the room needs it (lapel mics are best...