We narrowed to 1,042 results for: URE

...green cards and citizenship.   Lauren Celano, CEO Propel Careers. Lauren is passionate about working with...This post was contributed by guest bloggers Lauren Celano of Propel Careers and Rachel Casseus, Esq. ...and on. As you think about your career (and your future in general), keep in mind that you may have years...careers - being aware of this can help take the pressure off having to find the perfect job immediately...but not limited to biotechnology, chemistry, architecture, engineering, mathematics, physical sciences...bachelor's degree or its equivalent and state licensure, if required to practice in that field. H-1B work-authorization...background, expertise, publications, and plan for future activities; including the interest of the greater...

... 1) follows this structure: Step 1: fix (and maybe label) your sample (i.e. cultured cells or tissues...and after expansion and measure the degree of agreement between them to ensure the expanded images are...how you interpret a target’s structure. For example, cytoskeletal structures that form continuous lines ...easier, more accessible way to image biological features previously only visible via techniques like super-resolution...by just adding water.)  Step 5: Image   Figure 1: The ExM process allows you to physically magnify...depend upon the specific gel recipe used, ambient temperatures, incubation times, and the physical properties...properties of the sample itself. For example, cultured cells are typically easier to expand than mouse brain...

...survive high temperatures (up to 95 °C) and is active over a range of elevated temperatures (37–75 °C)....elevated temperatures, allowing further discrimination by incubating the ligation at a temperature near the...Reaction Temperature Calculator can be used to estimate an appropriate incubation temperature when using...will also efficiently ligate many undesirable structures, including substrates containing gaps or DNA ...phosphorylated base can be observed in the crystal structures of several DNA ligases bound to substrate (23... the melting temperature (Tm) of the DNA strands. This selectively reduces the concentration of annealed.... 2. Tomkinson, Alan E., et al. "DNA ligases: structure, reaction mechanism, and function." Chemical reviews...

....  After this, lowering the temperature allows the repressor to re-nature and again repress expression...red recombineering system has three components (Figure 1): 1) Exo, 2) Beta, and 3) Gam. All three are ...recombineering with an ssDNA oligo substrate.   Figure 1: Components of the Lambda Red Recombineering ...a generic lambda red recombineering experiment (Figure 2). In the following sections, key steps that differ...confirmation of recombinant clones.    Figure 2: Overview of using Lambda Red recombineering ... pairs of homology to the targeted insert site (Figure 2, top).  These primers are typically ~70 nucleotides...few strains can also be found at Addgene, but be sure to read the associated papers for their particular...

...copies per cell. Figure 1: A plasmid map showing the standard features of a plasmid. So, how...Growth conditions The amount of aeration, temperature, culture volume, antibiotic, and medium can all affect...chloramphenicol – make sure your growth conditions aren't working against you!  The culture inoculum Freshly...colony and do not subculture directly from glycerol stocks, agar stabs, or liquid cultures. Freshly transformed...pairs can be separated more readily at lower temperatures and allows the replication machinery room to...if you need two plasmids for an experiment, make sure they have "compatible" ori's. See the table below... affect copy number. Some ori's are temperature sensitive; others ori's can be "tricked" into amplifying...

... guest blogger Jack Leeming Editor of Naturejobs, part of Nature magazine. Humans are built to enjoy, ... your structure. Develop your protagonist In most cases, the protagonist will be you. Make sure to highlight...accept and acknowledge your failures. Foster conversation A story is not a lecture. Make yourself open to ... parts of our lives that tend to have a story structure: beginning, end; cause, effect; lesson, learning...apologised and admitted he’d been under a lot of pressure recently which had affected his judgement. Eventually...rhetorical technique you have in your arsenal to make sure people pay attention. Highlight your lowest points... discovery into a scientific story, but it will ensure that people pay attention. Practice Nobody gets...

...able to eliminate the need for axenic (pure) laboratory cultures to study these microbes. From 16S rRNA...GPS coordinates, water temperature readings using a heat gun, and pH measurements. We collected small water...samples could be kept frozen after collection. Figure 1: Paul included a voltmeter on top of the battery... bank and the vehicle battery/alternator to make sure the vehicle battery didn't run down. In September...processing environmental samples Since the high temperatures and unstable ground around these hot springs...-term stabilization of unknown bacteria. Figure 2: Christina Mork collecting samples at the hot...able to safely get back to our lab work. Figure 3: When a bison wanders into the campsite, leave...

... Staphylococcus aureus Cas9. Nature 520:186–191 . https://doi.org/10.1038/nature14299 Find plasmids from...targeting with CRISPR–Cas13. Nature 550:280–284 . https://doi.org/10.1038/nature24049 Find plasmids from this...altered PAM specificities. Nature 523:481–485 . https://doi.org/10.1038/nature14592 Find plasmids from this...and high DNA specificity. Nature 556:57–63 . https://doi.org/10.1038/nature26155 Ran FA, Cong L, Yan WX,...Streptococcus pyogenes (Sp) 3' NGG Staphylococcus aureus (Sa) NGRRT or NGRRN Neisseria meningitidis...than SpCas9. For example, Cas9 from Staphylococcus aureus (SaCas9) is about 1 kilobase smaller than SpCas9...more about SaCas9 and AAV Check out our CRISPR Featured Topic Page Resources on Addgene.org Check out...

...preparations are added to cells in culture. Three to five days later, cell cultures are inspected for sterility...also performed by ddPCR.  Quality Control Addgene ensures high quality viral vectors by optimizing and standardizing...quantity of gram-negative bacterial endotoxin is ensured to be less than five endotoxin units per mL. The...determine purity of the AAV preparation. Figure 1: Silver staining of purified and non-purified...item (see maps section for images).     Figure 2: AAV Pro cells were transduced with either pAAV-Ef1a-mCherry-IRES-Cre... darker than full vector particles.     Figure 3: Electron micrograph of AAV vector preparation...packaging system developed by the Trono lab. Cell culture medium (OptiPro SFM) containing lentivirus is first...

...developing transgenic hairy root lines in tissue cultures takes half a year, and generating a transgenic...in the medicinal plant, the Catharanthus roseus (Figure 1). The ultimate goal of our research is to enhance...production either in the plant or in plant tissue cultures.   C. roseus is not a model organism such as Arabidopsis...used to develop stable hairy root lines in tissue culture. This method is commonly used in C. roseus, but...not reproducible. Prior to creating transgenic cultures or plants, our transient expression method enables...overexpress a gene and evaluate its function. In nature Agrobacterium contains a plasmid with a T-DNA (... does not work well, as the leaves have a waxy texture and are difficult to infiltrate. We have recently...

...There is no known safe level of exposure to asbestos – i.e., any exposure to asbestos is potentially capable...the exposure, the greater the risk of disease) there is no known safe threshold or “safe” exposure level... goal of the Cancer Moonshot Initiative being to cure cancer, more funding and research opportunities ...unfortunately, completely preventable. Mainly caused by exposure to asbestos, mesothelioma is a form of cancer ... not quick. It can take decades from the first exposure to asbestos to the time when someone starts seeing...average latency period (time from first asbestos exposure to the time of diagnosis) is about 35 years, but... has noted, there is no safe level of asbestos exposure. Anyone exposed is at risk of developing mesothelioma...

... different reasons. Exposure to antibacterial drugs provides a selection pressure, killing off bacteria...resistance is defined as microbial survival despite exposure to antibiotics designed to kill them or to impede...multi-drug resistance: definitions vary in the literature, but generally organisms with multi-drug resistance...contributing to the development of resistance include (Figure 1): Over-prescription of antibiotics (for example...populations to antibiotics at unnecessary times, putting pressure on resistant bacteria to over-take the population... and making them more difficult to treat in the future. Patients not finishing the whole course of antibiotics...killed by a higher concentration, or sometimes more exposure time is needed for the drug to work… if the instructions...

...samples: What we did To measure the pH of our samples, we used a solution of cell culture medium, which is buffered...samples at room temperature and used the color of the liquid solution and pH paper to measure the pH. We compared...range of pH exposures. They showed that AAV infectivity was not altered by a short 2-hour exposure to pH as...-rings are, in fact, permeable to CO2 at low temperatures (-80°C) and once in the tube, the CO2 can alter...not all O-rings) were permeable to CO2 at low temperatures. We tested two different types of tubes: the...tubes after the indicated incubation periods, and measured the pH. We found that tubes subjected to a recovery... 2014). They also showed that even 24 hours of exposure to pH 5.1 did not alter the infectivity of the...

...pull-down assay, wherein a protein of interest can be captured along with its associated partners. Common pull-down...using antibodies against the target protein. The captured protein complexes are then analysed by mass spectrometry...contains the tag sequence flanked by homology arms (Figure 1). In the first step of optimization, we started...protein to form the ribonucleoprotein complex (csRNP, Figure 1). We use in-house purified Cas9 protein, which...using standard immunocytochemistry (ICC) assays.  Figure 1: Three ingredients of epitope-tagging experiments...RNA (IVT sgRNA) approach (Dewari et al. 2018 and Figure 2). Not only is the cr:tracrRNA approach highly...vectors or synthesize/purify IVT sgRNA in the lab. Figure 2: Cas9 RNP complexed with 2-part cr:tracrRNA tags...

... the off-target activity. Figure 2: Specificity is the measure of off-target activity (left) while...sensitivity. Site accessibility Site accessibility is a measure of which genomic sequences can be targeted by the... sites that were previously unavailable. Figure 1: The PAM sequence that the Cas nuclease recognizes...closely related to specificity. Sensitivity is a measure of the on-target activity of the Cas9 nuclease....frequency at a target site. A specificity ratio can be measured by calculating the on-target activity divided ...while sensitivity is the measure of on-target activity (right). Cas9 nucleases that exhibit enhanced...NGAN, NGNG, NGAG, and NGCG). Since then, the Osamu Nureki Lab described a rationally engineered Cas9, SpCas9...

... adjustments I wanted to make to ensure smooth filming in the future, but we were finally refining the...I could be happy with it. Cycle 5 - The Future! Figure 5: The mobile monitor and teleprompter being...trying all over again. Here’s that process!   Figure 1: The Design-Build-Test-Learn cycle adapted for...anticipated, we had to think of something else.   Figure 2: Ironing boards. Good as a WFH desk, and as a...important for me to let people record at their leisure. For virtual B-Roll (that’s the secondary footage...drawing board.  Cycle 3: A Return to HQ Figure 3: Even Blugene is practicing social distancing... left the room. Microphone: Lastly, I needed to figure out a microphone situation. Generally, I record...

...extension can usually ensure that the 3’-base(s) do not form secondary structures. The introduction (or...overlap ensures that the PCR generates a nicked circle rather than a linear product (see figure). Template... simply following the polymerase manufacturers’ PCR protocol. Be sure to adjust the extension time to ...vivo to give the final circular plasmid product pictured here. B) Screening of recovered plasmids for the...final concentration). The DMSO reduces secondary structures of the DNA template, and may also decrease the...template for site directed mutagenesis.  Polymerase To ensure that you don’t introduce undesired mutations through...polymerases such as Taq generate A-overhangs (a feature used in TA-cloning). Such non-complementary bases...

...Staphylococcus aureus Cas9. Nature. 2015 Apr 9;520(7546);186-91. doi: 10.1038/nature14299. Epub 2015 Apr...some research in human embryos. Nature. 2015 Dec 3. doi:10.1038/nature.2015.18947 5. Siva K, Covello G... mouse model, an important proof of concept for future preclinical and clinical work. Find Plasmids for... is of course much more difficult than in cell culture, but Ran et al. have shown that CRISPR and AAV ...homology-directed repair (HDR) pathway is downregulated in mature tissues - no problem, as exon skipping proceeds...would be applicable to 80% of DMD patients. Figure 1: Exon skipping strategies for Duchenne muscular...low-frequency indels will not cause frameshifts. Figure adapted from Osterout et al. (2015b) under a CC-BY...

... like pipets, or mini-centrifuges, etc. Be sure to capture as many of these tools as possible, this even... we suggest estimating the time it will take to secure your first (R-level) research grant.  Set your ...down prices as you create your list. This helps to ensure that all of the resources are accounted for. Write...an idea of how much you can expect to use in the future. 3. Identify the skills needed to use these tools...the talent pool that you’ll need to tap for your future research team. For example, if you’re going to ...help you define salary costs. 4. Ascribe a cost structure associated to these tools, skills, and personnel...searching for a faculty position. This is important to ensure that you’re looking at institutes that have the...

...to define what features would allow for the best performance in the most contexts (Figure 1). Together ...quickly losing activity. I set out to collect the features that would define a good tool for sticking together...Together the features with optimal biological performance are described as Click Biology (Howarth, 2025). ... Figure 1: Modular assembly tools, alongside criteria for Click Biology. Created with BioRender.com...bonds. Reactions also can happen at both body temperature and on ice, when we have cracked open the cells...genetically engineered components, like protein tags (Figure 2). Through the work of many groups, there are ...found in Addgene's repository (Table 1). Figure 2: Adding function to My Favorite Protein (MFP)...

...action potential (Figure 1). This light-induced excitation can take place in a tissue culture dish but can ... which often forces scientists to use invasive measures to perform in vivo optogenetic experiments. Intracranial...implantation of fiber-optic cables into the brain. Figure 1: With no blue light, the channelrhodopsin remains...improved photocurrent properties: The sequence and structures that determine membrane expression and plasma... electrophysiology, a technique that’s used to measure electric current of ion channels in living cells...electrophysiology. All thirty were functional and their measured photocurrent amplitude, wavelength sensitivity...were then characterized further by expression in cultured neurons and mouse brain slices collected following...

... Design and testing of eSpCas9 Looking at the structure of the Cas9 nuclease (PDB ID: 4OO8 and 4UN3), ...Interactions between the target strand and the gRNA (see figure below) Because off-target sequences have less ...theoretically decrease off-target cutting. Figure 1: Strand separation and target DNA binding to ...improve the specificity of Cas9 derived from S. aureus. This means you can likely apply similar rational...of genomic sites. Knowledge of Cas9’s dynamic structure leads to the development of HypaCas9 Researchers..., Chen et al. 2017 used single molecule FRET measurements to determine which portions of Cas9 might be...HypaCas9 plasmids are available from Addgene here! Future possibilities for Cas proteins With their enhanced...

...bacterial culture into the late stationary phase. Growing the culture at a lower temperature such as 30...  Figure 3. Summary of plasmid multimerization troubleshooting tips. Portions of this figure were ... More research is ongoing to determine plasmid features that affect the frequency of multimerization. .... Theoretically, as long as all the plasmid features remain intact, plasmid multimers function just as...recA-independent mechanisms. Avoid overgrowing the bacterial culture Another way to prevent plasmid multimerization ...optimizing the incubation time can help maintain cultures in log phase growth. In a study by Williams et...plasmid can exist in a bacterial population as a mixture of monomers and various forms of multimers (Summers...

...experiments require positive and negative controls to ensure proper interpretation of results. A positive control... to compare the experimental group.     Figure 1: A, Most biological experiments will benefit ...group and C, technical replicates or repeated measurements will increase the power of the results. In this...experiment. Technical replicates are repeated measurements of the same sample and are included to assess...long as the protocol remains the same.    Figure 2: Microscopy-based antibody experiments should...molecules through the interactions of other antibody structures, such as the Fc region. Fc-mediated binding interactions...as with an isotype-matched control.      Figure 3: A preimmune serum control addresses situations...

...p300-P2A-PuroR for lentiviral expression. Figure 1: dCas9-p300 adds H3K27ac marks to histones in...plasmid form or as ready-to-use lentivirus. Figure 2: dCas9-Tet1 demethylates cytosines at promoters...also available as ready-to-use lentivirus. Figure 3: dCas9-DNMT3A methylates cytosines at promoters...LSD1 to the smaller nuclease-dead Staphylococcus aureus Cas9 (Sa-Cas) and deposited plasmid IF311: pMAGIC...silencing was transient and quickly reversed in culture. However, DNMT3A-induced methylation persisted ...period, as this mark was faithfully propagated in culture and in vivo. In certain cases, epigenetic modifiers...acetyltransferase activates genes from promoters and enhancers. Nature Biotechnology 33:510–517 . https://doi.org/10.1038...

...polysaccharide structures, many of which are easily varied by the cell and whose structures are just beginning...around 8-12 h at room temperature as opposed to overnight at optimal growth temperature. If you’re lucky enough...traction lately (1, 2), depends on knowledge of the structure(s) a given phage will recognize on a host cell... for by manipulating the nebulization time and pressure. Nebulization units, which can be purchased cheaply...attached with a hose to a nitrogen gas tank with a pressure regulator. Other methods for physical shearing...some clones being outcompeted by others in liquid culture, dilute and plate the complete library of transformants...paper (colony-side up) and leave for 1 h at room temperature. After this, it is helpful to subject your membrane...

...everything was more structured and controlled. I worked from Standard Operating Procedures (SOP’s) and every...perfect duration for me because it was sufficient to figure out if my image of lab work lived up to reality..., but in research you also experience a lot of failure and problem solving. I believe that is something...they never really prepare you for the amount of failure and problem solving scientists experience during...in ways that people from another background or culture could understand. I did, however, find it very ...learn how to cope with a different climate and culture, and also needed to be resourceful and adapt. By...doing experiments  without the constant fear of failure because I knew the tests worked and would produce...

...My name is Lauren Flynn and I am currently wrapping up my co-op with Addgene as a DNA Sequencing Analyst... member of the Quality Control team, I help make sure the plasmids that go out our doors are correct. ...minor, it’s time for me to start considering my future path. Talking to Addgenies has helped me get a ...concepts that are heard often during undergrad. Lauren Flynn is the Addgenie behind our "Meet an Addgenie...years ago at a CRISPR meeting.Read his Instagram feature here. You also don’t have to go back to school...that if you don’t get your PhD you’re set up for failure. Listening to Kenkel’s story has helped me realize...with our resident dinosaur! Read her Instagram feature here. One of our QC Scientists, Shreya Vedantam...

...through the addition of Cre recombinase (Figure 2). Figure 2: A knockout targeting vector designed...difficult to target specifically with a gRNA). Be sure to keep these techniques in mind when choosing a... to knock out exon 2 of a given gene.   Figure 1: A knockout targeting vector designed to insert...for targeting constructs. In the example shown in Figure 1, large sequences corresponding to exons 1 and...-eGFP-PGK-Puro from the Jaenisch lab.   Figure 3: A knockout/knock-in targeting vector designed... and the targeted exon in the gene of interest (Figure 4). When recombination occurs, the gene still functions...cassette has been placed into an intron.    Figure 4: A targeting vector to create a floxed allele...

...long dsRNA via an RNase III enzyme named Dicer (Figure 1). The plant kingdom has expanded its repertoire...involved in the biogenesis of different small RNAs (Figure 1). These siRNAs can exert their gene silencing... modifications of complementary gene sequences (Figure 1). As a result, gene silencing can be easily induced...from the ease of use in dsRNA applications. Figure 1: Schematic of an RNAi-inducing transgene. Typically...that facilitates the formation of a hairpin RNA structure, resulting in processing by the endogenous RNAi...greatest advantage of RNAi is the straightforward nature of the technique—all that is required dsRNA complementary...complementary to your gene target as inverted repeats (Figure 1). With this technique, multiple genes can be ...

...fusion proteins can visualize: Cell Structure Plasmids: Cell Structure Plasmids: ER mCherry-Sec61 ...domain. Feng Zhang's group utilized the bi-lobed architecture of Cas9 to engineer a series of "split" cas9...for iLID nano and 800nM to 47µM for iLID micro; Figure 1). With this great affinity range, these LIDs ...light-mediated subcellular localization in mammalian cell culture and reversible control of signalling pathways. ... off and on your favourite signalling pathways (Figure 2). They are all available now at Addgene. 1...efficiency and gene expression. The pUCXKT vector ensures that all screened plasmids contain a gene variant....g. the ER) and a reporter protein of interest (Figure A). Franck Perez’s lab has engineered hook proteins...

...halt degradative processes while maintaining the structure of your samples, the relationships between cellular...that occurs depends on the incubation time and temperature, so you may be able to recover good antibody ...cross-linking, but it doesn’t preserve sample structure quite as well as a cross-linker. This is because...fixation with these chemicals, which impacts sample structure and could also cause your target to be washed ...Furthermore, these chemicals can also alter the structure of proteins, which could disrupt your target epitope...good thing depending on your experiment.   Figure 1: Choice of fixation method impacts antibody binding...dehydrates the protein, which can disrupt protein structure and thus structural epitopes, but can reveal linear...

...vectors effectively. Retroviral structure   Figure 2: Structure of a retrovirus. Image re-used ... you can use Figure 1 to compare the genomes of these two family members.   Figure 1: Gamma-retrovirus...2021 under CC-BY license. Figure 2 gives a detailed view of the structure of a retroviral particle, which...   Figure 3: The life cycle of a retrovirus. Image courtesy of Jeremy Luban, MD. Figure 3 depicts...vectors correctly. A solid understanding of the structure of a wild-type viral particle — and of the normal...depicts the viral life cycle. One should ponder this figure often, especially the three steps from the 12 o...

...bacterial recombination pathway to allow any cloning procedure to be performed using a simple two-step, 2 hr ...hr protocol prior to transformation (Figure 1). As this approach requires no special reagents or purification...be immediately adopted by any laboratory. All procedures are performed using the same three step protocol...cells. Primer design Using specific primer design (Figure 2), IVA cloning can be used to perform any plasmid...design for each type of modification is shown in Figure 2, and is discussed in more detail below. The main... the template binding region, with a melting temperature (Tm) of around 60 °C for all primers. The length...assembly. Combining multiple modifications A key feature of IVA cloning is the ability to combine multiple...

...fluorophore at the respective subcellular structure (Figure 1). Here, MXS Chaining gave researchers the...various mammalian promoters), the same cloning procedure can be used to incorporate each element into the...These fluorescent reporters can be used to detect structure and protein localization, as well as cellular ...with MXS-chaining were used to label subcellular structures in live cells. Four individual expression constructs...fluorophore, and thus enabled labeling of particular structures (Table 1). Each construct was flanked with a ...final construct was then introduced to HeLa cells (Figure 1). The resulting HeLa cells show robust labeling...Table 1: MXS Constructs for Labeling Cellular Structures Construct Fluorophore Excitation/emission...

...toolbox. We'll cover how Cas13a was identified, the structure and function of Cas13a with a focus on what makes...genes that fell into 3 categories based on the architecture of the CRISPR protein in question: C2c1, C2c2... referred to as Cas13a) is unique in terms of structure and function, and will therefore be the focus ...cleaves RNA rather than DNA substrates. In terms of structure, Cas13a shares no homology to the most commonly...CRISPR Blog Posts Applications of Cas13a   Figure 1: Using Cas13a as a diagnostic tool. A pool of...applications of Cas13a given what we know about its structure and function? For starters, Cas13a can be used...2017). This system, dubbed SHERLOCK (depicted in figure 1) has been used to differentiate strains of Zika...

...Zhang, a Ph.D. candidate in the Yang Lab, Qi Li, a lecturer at Sichuan Normal University, Jieze Zhang, an ...plasmid that expresses the S. pyogenes Cas9, a temperature-sensitive replicon for self-curing, an arabinose... now take place. After editing, pTargetF can be cured from the cell by inducing expression of the sgRNA...other edits. Once you have all the edits you want, cure pCas9 by growing the cells at 37 °C.     The new...us to update the pCas/pTargetF system.   Figure 1: The editing process begins with the addition...DNA for editing to take place. Then plasmids are cured from the cell. Steps 1-1 and 1-2 can be done simultaneously..., 2) changed the replicon of pCas into a non-temperature-sensitive replicon, and 3) added the sacB gene...

...required longer exposure time than its protein equivalent and was less stable due its RNA nature, it paved ...including: Environments with higher temperatures, since proteins can denature while aptamers are more stable...form the base of the stem in an RNA stem-loop structure (see Fig. 1a). When the aptamer binds its ligand...laboratory took advantage of their deep knowledge of the nature of GFP fluorescence to create an improved fluorescent...fluorescent aptamer system. Knowing that GFP’s  structure stabilizes a 4-hydroxybenzylidene imidazolinone...cells. The new aptamer also has a higher melting temperature than its Spinach ancestors, and has a lower magnesium... its biosensor capabilities (see Fig. 1b). The future of aptamer fluorophores The Jaffrey lab and others...

.... These features enable the fibers to emit light for optogenetic purposes, measure temperature, study ... described, then measured electrophysiological potential afterwards. This measurement trended with expression... the fibers would need to be, as well as other features necessary for implantation in brain tissue. The...the wild west for fiber implants. Due to the soft nature of the intestines and the requirement for digestive...multiple channels and can also record tissue temperature proximal to the implanted fibers. As important...Alternatively, the system could be used to deliver or measure new variables. The sky is really the limit for ...wireless modulation of gut and brain neural circuits. Nature Biotechnology, 1–13. https://doi.org/10.1038/s41587...

...amplification and verification processes we use to ensure high-quality library distribution. You can also...library contains many plasmids, care must be taken to ensure that all of these plasmids are amplified and none...First, use enough DNA and competent bacteria to ensure all components of the library are represented. ...performing amplifications, and we use electroporation to ensure a good transformation efficiency. For a typical...Addgene still recommends plating the transformation mixture on solid media. The colonies are then scraped off...we created for the Brunello library is shown in Figure 2. You can use a small PCR product because the ...AUC of 0.5 representing perfect uniformity (see Figure 3A). Addgene finds that this representation is ...

...experimental settings: HEK293 cell culture and in live zebrafish. For cell culture experiments, only dL5**-expressing...just 1 minute of exposure. MG-2I-dL5** experienced minor bleaching after longer exposure times. Additionally... fluorogen that can be used to ablate cells in culture and in vivo.  Read on to learn more about this ...exposed to light stained dead within 30 minutes of exposure. 90 seconds of illumination were sufficient for...mCer3 cells present 96 hours post treatment (see figure 2). Induced cell death was confirmed by TUNEL staining...light. They found that KillerRed required longer exposure times (5 minutes vs 10 seconds) to achieve the...genetically targetable near-infrared photosensitizer. Nature Methods,13(3), 263-268. doi:10.1038/nmeth.3735....

...prior to your departure for the new placement. Make sure you read any relevant literature for the techniques... have structured visiting positions within defined labs which you can apply to. These structured types...institute and mentor that they may want to consider for future work placements. The chance to learn and collaborate...prior to arrival so that you can work together to ensure your expectations are reasonable, your reagents...Unless some of these logistics are covered by a structured visiting program, you will have to make arrangements...get the ball rolling! If you are applying to a structured program, they likely have guidelines to consult...don’t make your bench mentor wait on you!), make sure you are prepared (read any protocols the day before...

...possible — and may contribute to more in the future. Figure 1: A timeline of CRISPR base editing advances...without DNA cleavage. Nature, 551(7681), 464–471. https://doi.org/10.1038/nature24644  Gaudelli, N. M., Lam...double-stranded DNA cleavage. Nature, 533(7603), 420–424. https://doi.org/10.1038/nature17946  Lander, E. S. (2016...receive a liver transplant — creating intense time pressure for the research team. Ultimately, the lifesaving...off-target editing. Base editing in the present—and future The final base editor that was administered to ...increased activity and therapeutic application. Nature Biotechnology, 38(7), 892–900. https://doi.org/...improved Cas domain compatibility and activity. Nature Biotechnology, 38(7), 883–891. https://doi.org/...

... conjugated antibody. Compensation beads are a mixture between synthetic beads that can bind to a conjugated... your voltage using your single color controls (Figure 1A), instead of using your actual samples. They...the settings with experimental samples. Figure 1: Example flow plots for a single color control... may otherwise be difficult to separate out. In Figure 2, you can see that, without the FMO, it would ...population from the Ly6C positive population. Figure 2: An FMO gating example. The FMO sample (A) can...clear positive and negative population. If you’re unsure what your population will look like for a particular...provided by the antibody companies or check the literature. Pro tip! Early gates, like your live/dead markers...

...yeast, are temperature sensitive screens. These involve raising or lowering the culture temperature to exacerbate...Selections do require there to be a culture condition that can apply enough pressure to select for variants of ...individually administered and then outcomes are measured. While the entire genome can be knocked out with... have a screen that doesn’t yield any hits. Make sure you plan ahead and consider these questions to save...though. A selection can take the form of varied temperature, nutrient availability, drugs, and much more....with screens. Selections require there to be a culture condition which eliminates manipulations that do...conditions which convey resistance to a drug. You could culture cells in the drug of interest at a lethal dose ...

...perturbation The Penn State Protein Ladder Engineered miniature CRISPR-Cas system for mammalian genome regulation...frankenbodies include mEGFP, mCherry, and HaloTag. Figure 1: (a) Anti-FLAG frankenbody (FB) construction ...advantageous to Cas9’s larger expression requirements (Figure 1), but Cas12a has generally been underutilized...finding acceptable variations in DR sequences. Figure 2: comparison of Cas 9 vs. Cas 12a expression cassettes...optimized toolkit furthers Cas12a’s potential in future genetic screens.   DeWeirdt PC, et al. Nat Biotechnol...commercial ladders which also provides a useful yet low-pressure teaching opportunity for lab members or students... to protein expression and purification. Figure 3: The Penn State Ladder run on SDS-Page gels. ...

... from viruses that infect people in nature, extra safety measures, like replication incompetent viral ...the stomach flu), and many more. Their infectious nature allows them to easily deliver their “cargo” to ... make you sick…well, you feel it. But the same features that make these familiar viruses such a pain is...biological tools.   Viruses as biological tools In nature, viruses infect cells and organisms (which we refer...of cell the virus will target and fuse with. In nature, this determines which organisms and cells they...Institute. Its genome, however, isn’t enough to ensure the virus can replicate and spread. Instead, viruses...are required. We will be covering these safety measures in a separate, upcoming post. If a viral vector...

...(rAAV) vector (Figure 1). Everything else outside the ITRs gets left behind.    Figure 1: Recombinant...polyA signal. Created with biorender.com. ITR structure  WT ITRs are symmetrical 145-nucleotide sequences...palindromic arms (A-A’, B-B’, and C-C’) that give the feature its characteristic T-shape. The arrangement of ... the ITR, which can either be “flip” or “flop” (Figure 2). Additionally, the ITR contains a 4-nucleotide...target site for Rep proteins (Savvy et al. 2013). Figure 2: AAV2 wild type ITR in flip and flop orientation... B-B’, and C-C’ regions) and forms a T-shaped structure. The Rep binding element (RBE) and terminal resolution...FAQs Due to their high GC content and hairpin structures, ITRs are prone to replication errors and disruptive...

...repository, but be sure to let us know if you'd like to write about your plasmids in a future blog post. No...under the control of the cI857 temperature-sensitive repressor. The temperature-controlled expression of the...crystallographic structures available in the PDB. Parton DL, Hanson SM, Rodríguez-Laureano L, Albanese SK...articles. These brief articles highlight the main features and applications of a partiular plasmid or set...arduous. The cloning process alone involves much literature searching, DNA synthesis, and plasmid assembly.... The pooled library, designed to be used with cultured Drosophila cells, targets 13,501 fly genes with...will create new traits in bacteria. Wang, et al. Nature. 2009. PubMed PMID: 19633652 Court, et al. Annu...

...and one NHEJ-repaired indel, often occur. Figure 1: In conventional gene targeting (A), you must...be included in the final ssDNA template. Figure 2: Overview of ivTRT to generate an ssDNA repair...challenging for sequences with complex secondary structure. Today, multiple companies offer long single-stranded...directs cleavage right before the stop codon!  Figure 3: How to design a C-terminal insertion with Easi-CRISPR... To avoid re-cutting by Cas9 after editing, make sure that the repair template does not contain the wild...facing towards or away from each other.  Figure 4: How to design a conditional allele with Easi-CRISPR...you have a plasmid with your desired insertion feature (e.g., a fluorescent protein), you can amplify ...