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...are designed to be complementary, but each primer may extend beyond the complementary region as long as...products (green) are digested with enzyme “A” as compared to the products of parental plasmid digestion ... Primer Design As a rule of thumb, 11 bp of complementary sequence on either side of the desired mutation...template should be isolated from a methylation competent bacterial strain (e.g. DH5α which is dam+). Methylation...A-overhangs (a feature used in TA-cloning). Such non-complementary bases at the 3’-end interfere with plasmid reconstitution...that any residual parental plasmid (usually from incomplete DpnI digest) can also form colonies under these...

...5). Optimization of this system defined three components required for translation (Figure 1): A tRNA ...the pSer-incorporated polypeptide. These three components were shown to effectively incorporate phosphoserine... the phosphoserine OTS, Sep-OTS, contains the components required for the translation of pSer-incorporated...host strain for pSer-incorporation The second component necessary for phosphoserine incorporation is a...enzyme function, protein interaction, etc. by comparison to non-phosphorylated or ‘wild type’ control ...substitution. Alternatively, you can express the completely wild type protein from the same plasmid.  Upon...dephosphorylation reaction can be tracked through the comparison of phospho:non-phospho species on PhosTag gels...

...transform into competent cells. The master mix of enzymes can be purchased from a company (e.g. NEB or ...so the entire reaction takes an hour or less to complete at 50 °C. After an hour or so, the sample is immediately...master mix for 1 hour, and then transform into competent cells. The success rate of this reaction is typically...terminator sequence), since this would directly compete with the required single-stranded annealing and...ensures correct assembly order of the fragments as compared to the smaller overlapping sequences left with...assembly meets CRISPR Gibson can be adapted to more complicated cloning schemes, such as those where the vector...

...credit for what we have accomplished. Starting with a recap of accomplishments, from learning new techniques...tangible research and career goals, and celebrate accomplishments. These techniques are laid out in Vincent et...conversation. Goals and Planning Worksheet: Lists accomplishments, research/professional goals and feedback. ...including major project goals and effort needed to complete them. These documents dictate the subject matter...concrete plan in hand, trainees are motivated to complete their work rather than overwhelmed or confused...

...1]. Like other transposons, piggyBac™ has two components, a transposon and a transposase. The piggyBac...also enables the excision of the transposon in a completely seamless manner, leaving no sequences or mutations...enhanced random integration efficiencies that are accomplished with transfections and are therefore virus-free...enhance gene editing? Gene editing can now be accomplished in a more cost-effective and efficient manner...challenges still remain, such as making gene edits completely seamless (i.e., so no other genetic alterations...transposase (PBx), which contains mutations making it competent for excision, but defective for integration [4...   How does the piggyBac™ gene editing system compare with other methods of gene editing? There are primarily...

...services don’t offer plastic recycling, specialized companies can recycle plastic-- you just have to find them...product packaging) for free! It has to be from their company and it has to be packaging (no coated tissue culture... label. Recycle nitrile gloves Terracycle is a company that takes items not commonly accepted at recycling...your lab or department and send them back to the companies for reuse. They’re really easy to spot and even...recycling program at your institute! Universities and companies have the potential to make a huge impact directly...! These days, it’s important for individuals, companies, and universities to be sustainability leaders...

...methylation competent E. coli for this procedure to work properly. Directly transform chemically competent cells...tips for applying it in your own lab. Table 1: Comparison Between Standard Restriction Cloning and Fast...are then transformed directly into chemically competent E. coli and transformants selected with the appropriate...insert with an additional 15-17 bases that are complementary to the overlapping vector ends. See an overview...an hour at 37°C. This should be enough time to completely digest the template DNA. Note—templates must ...

...sequence. This means cloning occurs in vivo, as compared to restriction enzyme cloning where the genetic...homology to the target site for recombination. Key components of the Lambda Red recombineering system The lambda... lambda red recombineering system has three components (Figure 1): 1) Exo, 2) Beta, and 3) Gam. All three...dsDNA was short enough, a ssDNA whose entire complementary strand was degraded. Beta: Beta protects the...created by Exo and promotes its annealing to a complementary ssDNA target in the cell.  Only Beta expression...an ssDNA oligo substrate.   Figure 1: Components of the Lambda Red Recombineering system. See ...region of interest and design an oligo that is complementary to the lagging strand. In practice, it’s easier...

... of powdered media. Filtration does not ensure complete sterilization as mycoplasma can even escape 0.2... growth of mycoplasma but cannot eradicate it completely. When mycoplasmas are seen in the culture, it...mycoplasmas are used. A broad range of primers encompasses almost all the infecting mycoplasma. The primers...assay kits are available from different biotech companies which are enzyme-based mycoplasma detection kits...synthesized by eukaryotic cells. First, the kit components rupture the mycoplasma and then specific enzymes... of mycoplasma. These methods cannot guarantee complete removal of mycoplasma but, can get rid of most...Microbiol .44: 42–49  Uphoff Cc, Drexler Hg (2002) Comparative Antibiotic Eradication of Mycoplasma Infections...

...throughout your career. Plus, just one role can encompass many of these activities leading to a rich day-to-day...combat sexual harassment in academia or a large company who decides how they are going pursue regulatory...science policy roles exist. Explore these resources compiled by the AAAS Science and Technology Policy Fellowship...working in science policy doesn’t have to be a complete career pivot. There are many ways to pursue your...system? Defining your why is what will make you a competitive candidate for any science policy position and...expertise and then once selected, you can pivot to a completely new field without issue. This may be more challenging...much smoother.  Action Items:  Explore this comprehensive resource developed by Dr. Kelly Singel which...

... TF library (aka the MORF Collection) and an accompanying expression atlas, including over 3,500 human...from these sites. The Zhang lab generated a comprehensive library of each unique TF open reading frame...endoderm, and mesoderm). These profiles were then compared to that of reference cell types generated by the... the human fetal expression atlas. Computational analysis was used to infer differentiation trajectories...Previously engineered TF libraries were not as comprehensive as this one, so it is an invaluable tool to ...expression dysregulation event. Diseases are often complex, with multiple layers of dysregulation from either...

...that no component of the overall project gets left undone or is duplicated. With each component of the ...the project completed by an individual who is a local expert in that task (or has the bandwidth to become...result is high quality and the entire process is completed more efficiently. Listen to our podcast interview...desired outcomes. Concern yourself with what is accomplished, rather than detailing how the work should be...in more engaged, successful employees who can accomplish great things.   To read more on this topic and...

...fluorescent proteins, how to do a head-to-head comparison of fluorescent proteins and how to make a well-informed...your selection on a small-scale head-to-head comparative study under conditions that best mimic the intended...protein properties and provides a quick way to compare fluorescent proteins, e.g. see Chudakov et al (...is of no use. Therefore, it is more sensible to compare fluorescent proteins based on their practical brightness...promising fluorescent proteins and to determine and compare their practical brightness in a system closely ...application. In figure 2 we provide an example of the comparison of the practical brightness of two orange fluorescent...

...expressed in cells, generates much less signal than comparable fluorescent proteins. This weak emission hasn... with increased sensitivity and faster imaging compared to previous publications. They also developed ...they successfully imaged multiple subcellular compartments over several minutes using Nano-lantern fusion... fluorescence, so higher concentrations of the compound can be added to the media. Cellular esterase then...producing a constant supply of substrate. Using this compound to excite the Nano-lanterns, Takai et al. continuously... handy. This approach is especially suited to complement optogenetic tools, where light used for excitation...

...all bacteria are naturally competent to take up DNA, they can be made competent through chemical manipulation...Addgene’s blog to learn about making your own competent cells and our protocols page to learn about bacterial... The phagemids are packaged into replication-incompetent phage particles with assistance from a ‘helper... A., and Karen Guillemin. "Multiple phases of competence occur during the Helicobacter pylori growth cycle...Meibom, Karin L., et al. "Chitin induces natural competence in Vibrio cholerae." Science 310.5755 (2005):...

...and reduce needed compensation.    Working with antibodies can get a bit complicated, but hopefully this...time-sensitive, using precious samples, or being compared to other data in a manner that demands high accuracy... reaction to understand if any of your buffer components could affect the reaction. For instance, sodium...detected in the same assay. This is one of the most complex subjects in antibody-based protocols - but thankfully...another channel's detection range. Though you can compensate for this when reading your data, you’ll want ...

...track of computation work comes with new sets of challenges. One challenge with computational analyses... of biologists are finding themselves using computational approaches to analyze large data sets (myself...RNA-seq analysis. I had never interacted with a computer at the command line, and so was really starting...especially since you’ll want to dig in to the computation, but they will serve you well as you perform ...directory: A directory is a file system in the computer and is just another name for a folder. For instance...

...increased versatility in probe attachment, and compatibility with far-red and near-infrared fluorophores....enable highly specific visualization of cellular components, typically a protein of interest. Fluorescent...ligands Effects on protein function and cellular components: It's important to assess how dye-conjugation... function and its propensity to bind cellular components. Some dyes target specific organelles like mitochondria...may prove challenging to stain intracellular compartments in in vivo model systems, so extra care must...Transfer (FRET) applications, the dyes need to have compatible excitation and emission profiles to enable effective...finding the right dye for your experiment more complicated. We hope this post helps you in your search for...

...based on size. Whether higher molecular weight complexes are detected will depend on the homodimer affinity...During the experiment, the solution is diluted, complicating the interpretation. Because of this, these methods...fluorescent protein is monomeric without the accompanying data to prove it is worthless. The OSER assay...and biological properties of new fusions can be compared with mEGFP or mTurquoise2, which are established...that you will be using. If you do a head-to-head comparison right, it will be valuable information for the... are engineered can be shared to simplify the comparison by others. Together, we can build a valuable ...

...us to the key question: what is the best way to compare the photostability of different fluorescent proteins...measurements for different fluorescent proteins and by comparing how the fluorescence intensity changes over time...photostability of the fluorescent proteins can be directly compared. An example of a photostability measurement of...fluorescent protein can be identified by a head-to-head comparison of several fluorescent proteins of the same spectral.... In figure 1 we provide an example of such a comparison between cyan fluorescent proteins using widefield... power, the photostability of the variants is comparable (lower panel of figure 1). To avoid changes in...