We narrowed to 1,017 results for: PLE

...lympho-/splenocytes, you might only be interested in one of the various cell populations; for example, CD8...These methods can include labeling markers with multiple proteins/antibodies, targeting markers found within...beyond standard surface labeling. Pro tip! Many people use 'labeling' and 'staining' interchangeably to...antibody can originate from a range of species; for example, rabbit or mouse. In a second step, a fluorophore-conjugated...Otherwise, your secondary antibody will bind to multiple primary antibodies, yielding a false positive ...FACS achieves greater purity of your sorted cell sample than MACS. An effective strategy to sort out your...the permeabilization solution can be the same). Examples of such targets include transcription factors,...

...avidin can be added to your sample. Any free biotin-binding sites on this complex can bind to your biotinylated... proteome that are biotinylated. In E.coli for example, the BPL, BirA, is responsible for biotinylating...plague these animals even if their diet was being supplemented with biotin. Thus something in the egg white...discovered in the 1960s, strepavidin. The Biotin-avidin complex can then be used and/or manipulated for a scientist...immunoprecipitation experiments. Biotinylated proteins or complexes can be easily purified using columns or beads ... added to the reaction for easy detection. The multiple biotin binding sites found on avidin are also ...This creates a large meshwork of biotin-avidin complexes bound to your protein of interest leading to amplification...

...equipment, reagents, animals, glassware, and even people. When developing a budget, I suggest that you focus...consumables (such as pipet tips, glassware, etc), get a simple list from your current P.I., lab manager, or administrator...need to tap for your future research team. For example, if you’re going to need a tech because you’ll ...regions where the host institute is located. For example, salaries in Kansas versus salaries in NYC will... that have the means to support your work. For example, if your work requires specialized equipment, try...need.  Prioritizing resources in your budget The simple practice of putting together a resource budget ...rather than focusing on long term investments. For example, purchasing “used” equipment can save you money...

... shape what people know and believe about the world. It is vital to understand what people know – or, ...like?  One of my more recent studies looked at how people perceive and evaluate peer-reviewed sources of ...called the “biased evaluation of evidence,” in which people (conscious or unconsciously) pick apart sources... to dismiss them. There’s evidence to show that people with greater levels of science literacy are especially...hands-on science communication. Between New Zealand’s multiple Covid-19 lockdowns, I held mask-making workshops...the difference between airborne and aerosolised droplet transmission and how different fabrics and mask...illness. I later helped run workshops where I showed people how they can make reusable cloth menstrual pads...

...you need more than one?   Multiplex assays Multiplex assays involve multiple antibodies, each with a different... the antibodies time-sensitive, using precious samples, or being compared to other data in a manner that...this post, the conjugates, or signaling molecules! Please keep in mind that this is just a brief introduction...conjugate that can be activated to emit light with a simple kit, and is readily available on any number of ...ain’t broke, don’t fix it. HRP is easy to find, simple to use, not sensitive to light in its inactive ...detected in the same assay. This is one of the most complex subjects in antibody-based protocols - but thankfully... this is an Antibody 101 post, so we'll keep it simple. If you follow the two basic guidelines below, ...

...minipreps for small batches of samples. As soon as that process is complete, QC scientists prepare Sanger...mutation.  Once the samples arrive, each group of plates will have select samples prepared and screened...collection in your experiments and published your data, please fill out this form and we’ll add your citation ...available. Plasmids are continually added as they complete QC and you can find all available plasmids listed... high copy backbone with a T7 promoter driving multiple combinations of WT, codon optimized, untagged,...screened by NGS to spot check the samples, the rest will be put into a status we call “Archived.” This status...for all several thousand plasmids at once. Any samples that are requested by scientists, will immediately...

...cerevisiae yes - This can complement ura4- S. pombe, but the complementation is weak. yes   LYS2 L-lysine...no S. cerevisiae yes - This can complement leu1- S. pombe, but multiple copies are required. yes   TRP1...vectors? Yeast are eukaryotes and thus contain complex internal cell structures similar to those of plants...mechanism of homologous recombination to facilitate simple gene replacement/mutation. Yeast expression plasmids...selection markers.  The yeast origin of replication (Please note: This first section primarily pertains to ... including a copy of a functional gene which complements the host’s auxotrophy. When grown on media NOT...drug selection methods feasible in yeast as a complement or alternative to using auxotrophic markers. ...

...twice a week. I have done this with people I work with and people from outside my work for almost 20 years...they know some of the people you work with Give more informed feedback (for example, they may be able to... and way of expressing it in meetings? The more people you meet, the more potential mentors you will identify...tactics to try. One of my favorites is making the pledge to have lunch or coffee with someone I don’t know...tap them for advice. As a future alum, these are people you can “cold call” and ask for some of their time...friendships or work interactions. Watch for the people that are already teaching you and see if there ...have a broad range of mentors you will always have people you can turn to for help and advice. If you missed...

...with the same genetic background and do an apples-to-apples comparison of all those mutations. If you ...compares to his CRISPR/Cas9 system. Addgene: For people unfamiliar with this new TALEN kit, can you describe...especially well-suited for use in stem cells. (See the complete protocol at StemBook). Addgene: How are you now...particular variants or mutations have been reported in people, we can use this kit to knock in those mutations...disease process. In the Cell Stem Cell paper, for example, we generated hepatocytes to examine the effect...much all investigators. Our kit makes it extremely simple to design TALENs. It only takes two to three days... find several mutations in the same gene or in multiple genes, all of which seem to contribute to a particular...

... fragment of the NanoBiT® complex, which, upon binding to its complementary partner LgBiT, reconstitutes...disordered and structurally complex. The tumor suppressor TP53 (p53) is one example of these difficult-to-study...with nothing more than substrate addition and a simple add-mix-measure method. NanoBiT® luciferase activity...also be combined with the HiBiT tag to provide complementary validation of protein expression and localization...or co-cultured across very narrow filters, complementation in recipient cells proved that the enzyme was...and tissue processes, but their low abundance, complex regulation, and context-dependent activity can ...fluorescently-labeled VEGF-A to the heteromeric receptor complex. Find sHiBiT-KDR Fusion Vector at Addgene! HiBiT...

... The destination vector contains sites with complementary overhangs that direct assembly of the final ...Unique 4 base overhangs can be used to assemble multiple fragments - up to 10 fragments are commonly assembled...fragment in an inverse orientation. This allows for multiple DNA components (promoters, genes, terminators,... reaction. For detailed Golden Gate protocols, complete with helpful tips and tricks, see The Sainsbury...editing which allowed for the ordered assembly of multiple DNA fragments to create TAL effector nucleases...plasmid, but it can also be adapted to express multiple gRNAs. Addgene has two Golden Gate-based gRNA ... 7 gRNAs into one destination vector, making multiplexing easy.  Disadvantages of Golden Gate cloning...

... SCF E3 ubiquitin ligase complex. In the presence of auxin, this TIR1 complex binds to the degron sequence...elegant system that allows for the assembly of multiple DNA fragments efficiently and accurately in a ...a nucleus -> DNA compaction around histones -> Examples of histone-modifying enzymes and reader-effector...assembly (backbones available at Addgene) with examples of potential synthetic chromatin mediators and...system will allow investigators to rapidly create multiple synthetic chromatin-modifying proteins quickly...protein degradation and quantification of this depletion using imaging-based measurements (Ashley et al...of your protein interest would also result in depletion of the FP, which can be easily visualized.  The...

...sequence can completely disrupt expression of the transcribed ncRNA (Jiang et al. 2014).  Simple knock-out...possibly be made up of. The answer is… drum roll please… non-coding RNAs! You probably didn’t see that ... However, removing large lncRNA fragments with multiple gRNAs poses the risk of disrupting adjacent genes...transcription of the ncRNA (Hazan and Bester 2021). For example, inserting a polyadenylation signal downstream ...interactions between ncRNAs and adjacent genes are complex and not yet fully understood, so it is important...is not effective for your experiments, there are plenty of other CRISPR-based options that can target ncRNAs...variants may also be useful for targeting ncRNAs. For example, the Cas12a variant can cleave single-stranded ...

...optogenetic tool called PdCO.  This is an "optoGPCR" (coupled to Go, so it's an inhibitory opsin!) and due to...AAV5 Optogenetics Yizhar New viral service with multiple serotypes pAAV_EF1a-DIO-PdCO-mScarlet-ER-miniWPRE...AAV5 Optogenetics Yizhar New viral service with multiple serotypes pAAV_hSyn-PdCO-EGFP-WPRE AAV1, AAV5...AAV5 Optogenetics Yizhar New viral service with multiple serotypes pAAV-CaMKIIa(0.4)-eOPN3-mScarlet-WPRE...AAV5 Optogenetics Yizhar New viral service with multiple serotypes pAAV-CAG-FLEX-rc [Jaws-KGC-tdTomato-ER2...AAV1, AAV9 Biosensors Li New viral service with multiple serotypes pAAV-hSyn-GRAB_OTmut AAV9 Biosensors... AAV9, PHP.eB Controls Feng New viral service, multiple serotypes Looking for more viral vectors? ...

...Level-1). Other possible tools, lentiviruses for example, are not approved for use in vivo use at the University...Manitoba due to their Biosafety Level-2 or 2+. Please Note: Despite the fact that AAVs themselves are...you can see, AAVs of each serotype can transduce multiple tissues. So how do you choose the right serotype...hippocampus and cortex) within the mouse brain (4). An example from the cortex is outlined in the table below....thawing. Although more stable than other viruses, multiple freeze thaw cycles can still reduce AAV titer....is possible to use AAV constructs with self-complementing structure (scAAV) (7). As single stranded DNA...mapping of circuits In our lab, we use AAV-DREADD complexes to study neural function. Often the reporter proteins...

...techniques differ in important ways, but can also complement one another by providing both broad and specific...Mismatch Cleavage Assay Not Provided 5% Inexpensive, simple Low-throughput, low sensitivity Sanger Sequencing...investigator can be relatively certain that their samples don’t have off-target mutations if they remain ...the clinic. NGS-based methods provide the most complete information profile regarding putative off-target...counts making them fast and viable options for multiplex sequencing in many laboratories. Nevertheless,...sensitive as targeted amplicon sequencing. For example, GUIDE-seq seems to have a minimum detection limit...DSBs, doesn't require high read counts, fast multiplexing Requires delivery of dsODN (potentially toxic...

...relatively easily synthesized, must be produced using complex mixtures derived from cells or using live cells... expression of mammalian proteins that require multiple post-translation modifications for proper protein...systems Insect cells can also be used to produce complex eukaryotic proteins with the correct post-translational.... These systems enable high expression of very complex, glycosylated proteins that cannot be produced ...expression. Protein expression in bacteria is quite simple; DNA coding for your protein of interest is inserted...pET, pRSET, Gateway pDEST, and pBAD vectors for example. Protein expression from each of these vectors ... expression systems, including the majority of complex post-translational modifications. Extraction and...

...amajLime, cjBlue, meffBlue, aeBlue, amilCP, tsPurple, and gfasPurple) (Figure 2). Figure 2: Chromoprotein...expressed and gives the cell a magenta (amilCP) or purple (tsPurple) color. Though the toxin is integrated in ...). Eight years later, scientists isolated pink, purple, and blue chromoproteins isolated from reef-building...just by looking at the cells on the plate. For example, if your plasmid contains a gene encoding a blue... Figure 3: pTOX plasmids expressing amilCP and tsPurple genes. Image: Lazarus et al., 2019. One such... lab is the Waldor lab who used the amilCP and tsPurple chromoproteins to create negative selection markers... and Tse2 toxins, respectively, and encode the tsPurple chromoprotein.  Find the allelic exchange vectors...

... CRISPR samples. We invite you to jump in and try it out, or take a look at our live examples: experiment...adhere to the same principles listed here. It employs biological best practices. For example, it ranks guides...November, we began using it internally. Guiding principles behind CrispyCrunch While CrispyCrunch may evolve...you can depend on it to stick to a few guiding principles that we've followed since the beginning. It'...if desired. It works as a whole or in part. For example, you can analyze experiments designed outside CrispyCrunch...active guide. With these constraints in mind, we implemented the following algorithm for optimal mutation ...Francisco. Before starting his career in software, he completed a MSc in Psychology at McMaster University in ...

...some simple solutions available for researchers to deal with all of these problems. The simplest and best...Stop using EGFP/GFP for fusion proteins! Despite multiple studies in high profile journal articles, many...forming dimers in cells. Why should you care? Three simple ways a dimeric FP could ruin your day (and experiment...cell. The Kd of EGFP is 0.11 mM [2]. Following the simple logic outlined above, you might think EGFP is unlikely... a cell. Unfortunately, things are not quite so simple. Concentration is the number of molecules within...around that fusion protein is very high. Real world examples of these situations in cells are described below...order oligomers. Unfortunately, FP dimerization coupled with POI dimerization can lead to polymerization...

...create and simple to multiplex. Problems with FRET kinase reporters Difficult to Multiplex: FRET reporters...easy to multiplex. Their design allows flexible swapping of fluorescent proteins and multiplexing is simple...reporters are challenging to design and difficult to multiplex. The Covert Lab provides an alternative tool with...properties that limit which fluorophores can be multiplexed together. Challenging  to Design: FRET reporter...

...briefly boiling the sample before use or by directly adding a small volume of the sample to the PCR reaction...orientation-specific primers. Blunt end cloning is an example of when you might want to know the orientation ... insert and one primer anneals to the insert. A simple way to create this type of primer pair is to mix-and-match...  Primer Design Pros Cons Insert-Specific Simple "yes or no" test results Doesn't tell you the... few ways you can do this. If you are going to complete your colony PCR analysis in the same day, you ... product size on a gel   Now that your PCR is complete, it’s time to run the products on an agarose gel...and to add a loading dye with glycerol to your samples before pipetting them onto the gel. The figure ...

...process is simple -  no restriction, ligation or gel purification steps are required! Multiple fragment...highly efficient way to move DNA sequences into multiple vector systems. With the appropriate entry and... understand the process, we’ll walk through an example experiment where we might use Gateway cloning to...attL-entry vector. This fragment is inserted in a multiple cloning site (MCS) of an attL-containing entry...lentiviral expression, we could use a vector like pLenti CMV Puro DEST (w118-1) or the doxycycline-inducible... cloning You can use Gateway cloning to insert multiple DNA fragments into many vectors at once in the...plasmids available at Addgene, including the Frew Lab Multiple Lentiviral Expression Systems (MuLE) Kit, the ...

...needed to transform the raw sequencing data into a complete plasmid sequence that would be ready for our QC...inherent in Nextflow’s design, we were free to use multiple programming languages (Python, Perl, Java, R, ... adaptable property gave us the freedom to use multiple open-source sequence assembly tools at the appropriate...containers are they are designed to ‘contain,’ in a complete package, all of the pieces of software that depend...bioinformatics workflow in Nextflow Establishing a complete pipeline however, required more than just identifying...from the first process to the second process. For example, our bbduk process created trimmed FASTQ files ...sure we understood the concept of channels and implemented the input declaration correctly, the script block...

...lab often tests samples of new bacteria from anywhere they can find, whether the samples are from a rain...was safe to say the sample had their attention! They isolated DNA from the sample, amplified the CRISPR...phylogenetically classified as the more complex Class I systems that contain multiple subunits, but the more they ...Cas7-11 appeared to have derived from the more complex type III-A and III-B systems. Though it was composed...the domains into a single protein, shedding the complexity of its ancestors. They classified the system ...cleaving RNA at positions defined by the target:spacer duplex without detectable non-specific activity. Its major...

...ever like to write about a recent plasmid deposit please sign up here.  Here's what you'll find in this ... list of specific structures within cells. For example, to improve nuclear localization of fluorescent...fluorescent proteins, they found that adding a triple nuclear localization signal or including fusions with Histone...//doi.org/10.1101/160374 Nanobodies- there's a simple method for that! By Michelle Cronin The benefits...Kimiko Hara, and Kazuo Umemura have described a simple method which alleviates the inclusion body problem...problem and makes nanobody production in E.coli as simple as following a DNA miniprep protocol!  First, the...https://doi.org/10.1016/j.pep.2020.105607 A new multiplexed CRISPRi and CRISPRa system for human pluripotent...

...function in unrelated host species, even from completely different kingdoms of life. That’s convenient... for researchers who want to do experiments in complex eukaryotic cells, but would prefer to grow, clone...systems don’t always translate to eukaryotes. For example, beta-lactam antibiotics like penicillin and ampicillin...pathways shared by bacteria and eukaryotes. For example, puromycin inhibits protein synthesis across kingdoms...all depends on how the antibiotic works. Another example is Zeocin, a DNA intercalator, which affects all...selection markers can be used across species, for example, in both S. cerevisiae and E. coli. Auxotrophic...stable mammalian cell lines, but it isn’t common for simple transfection experiments. Reporters Finally, in...

...appropriate time—for example, the cell must have enough nutrients available to complete the entire round ...and regulation in bacteria Before looking at an example plasmid, let's review how E. coli normally replicate... must bind ATP. Then, the ATP-bound DnaA binds multiple specific sites within the ori called DnaA boxes...stringently controlled plasmids, replication is tightly coupled to the bacterial host's cell cycle, maintaining... stringently regulate plasmid replication. For example, the plasmid ColIb-P9 encodes the protein RepZ ...Inc, also encoded by the ColIb-P9 plasmid, is complementary to part of the sequence forming this pseudoknot...replication. At high copy numbers, RepA proteins form complexes between iterons on different plasmids in a process...

... models. In complex diseases, it can aid in generation of multiple edits or implementation of expression...change in pathology. If the disease pathology is complex, a model may be most effective. Once drug candidates... all diseases are fully characterized or have a simple pathology. In these cases, preliminary identification... – the dreaded ‘undruggable’ proteins – having multiple hits or an entire pathway of possibilities greatly...made this control step extremely accessible. For example, let’s say you identified gene target X for BRCA-deficient...candidates are selected.      CRISPR aids in multiple stages of the drug discovery pipeline.   ...

...gene. For example, they eliminate the expression of the gene (by CRISPR knockout, shRNA depletion, etc.) ...genes – if a gene functions at multiple stages in development, its depletion may result in lethality before...redundant genes – multiple genes which function in overlapping roles can mask the depletion of one another...modifying the gene of interest in any way. For example, if green fluorescent protein (GFP) was the reporter...monitors due to transcriptional fusion.   Examples of promoter, enhancer, and gene traps at a genomic...

...slowly becoming a convert to the principle that you can't motivate people to do things, you can only demotivate...Intelligence or to hone delegation skills, for example. So aside from a focus on removing obstacles, what...grow in skills. Managing a team is hard and most people have some learning they need to do before they ...most from your team. It is pretty clear that if people are mostly happy at work, they will work harder...problems to solve (I think this is true of most people, not just scientists) Are self-directed, but need... in a 5 part series on Introduction to Managing People for Scientists. Subscribe to the Addgene Blog to.... Good introductory reference books on managing people: Skills for New Managers by Morey Stettner Successful...

... takes an hour or less to complete at 50 °C. After an hour or so, the sample is immediately ready to transform...to design primers for Gibson Assembly. For a simple example of using Gibson assembly, imagine that you ...will go over some advantages, disadvantages, and examples of how scientists are using Gibson assembly to...assembly is well known for allowing easy assembly of multiple linear DNA fragments, but can also be used in ...biology, mainly because of the ease in assembling multiple fragments in one step with no scar sequences remaining...where no other methods were available. A second example of using Gibson Assembly along with CRISPR was ...Whether it is for routine cloning, assembly of multiple fragments, or synthetic biology, you should consider...

...hand. Records of plasmid construction were often incomplete or nonexistent. In the 21st century, many molecular...molecular biologists didn’t know the complete sequences or properties of the DNA molecules they were using... (HCI), and we have adhered rigorously to HCI principles. For every task, we envision what the user wants...refinements in response to customer feedback. An example of this approach is SnapGene’s algorithm for detecting...could annotate a feature inaccurately. The best example is fluorescent proteins, which often come in closely... harness common feature detection in a new way, please contact us at snapgene.com. Many thanks to our... She is passionate about building software that people love to use, and likes to start at the beginning...

...platform that allows us to provide scientists with complete plasmid sequences. After launching our viral service...accommodate adeno-associated viral vector (AAV) samples and created a simplified process, termed viral ...parallel, there is a very real risk of mixing up samples, serotypes, or both. Unfortunately, the standard...present SMRT sequencing only works for self-complementary AAVs and the TA-based ligation and tagmentation...molecules in the DNA extract associate with complementary strands, enough double-stranded species exist...Pacouret, 2017). Moreover, the sequencing data from multiple AAV lots can be run in parallel and provide serotype...adeno-associated virus particles containing complementary polynucleotide chains. J Virol 9(2): 394-396...

...shown in the image below. An example of Gram positive (purple) and Gram negative (red/pink) staining...the CV+, creating a complex within the peptidoglycan layer, staining the cell purple. When the alcohol ...thinner membrane. Procedure The stain is a pretty simple process. First, flood the heat-fixed smear with... Gram positive bacteria will be stained blue or purple, and gram negative bacteria will be stained red...the peptidoglycan layer, trapping the large dye complex created by the interaction of CV+ and I- in the...staining procedure, gram positive bacteria are stained purple or blue, and gram negative bacteria are stained...

...need to give everyone a heads up (for example, notifying people of an updated protocol), send an email...Google doc where people can make comments. At meetings, we just focus on the parts where people had questions...steps (see “action items” below). Get the right people in the room There are few experiences more frustrating...our all-company meetings short, informative, and pleasant. Email: Sometimes, the right meeting is no meeting... positive peer pressure to make sure everyone completes their assigned items to keep the project moving...happen “off-line” with just one or two relevant people. How often do you need to meet? Use trial and...

...experience. I enjoyed helping people and learning to explain concepts in ways that people from another background...This was particularly interesting as I had just completed my thesis on the study of e2Fb, which is a tumour...cancer and I loved the practical aspects of testing samples in the lab, but I really struggled with the large...strong point. Working in industry: virology On completing my MSc, I again had to look at what type of work...English speaking country with a strong economy and plenty of jobs in science at world renowned institutes...loved the fact that my work had a real impact  people’s lives and would improve their treatment outcomes...of a dream job, but I knew that I enjoy helping people and decided to compromise and applied anyway. I...

...𝛽-galactosidase. Complementing this mutation by expressing this deleted portion, for example on a plasmid,...fewer colonies after your experiments. As a common example, a selection will leave you with the colonies that...plasmid contains your insert of interest or not. For example, choose enzymes that only cut once on either side...you will just see one band. These are only a few examples of how you can use restriction digest to screen...get started, you will first need a primer that complements your plasmid sequence. Start with a backbone-...backbone-specific primer that will sequence over the Multiple Cloning Site (MCS) and into your insert. Check ...can confirm the sequence of the entire plasmid. Please note that NGS verification is more time-intensive...

...scientists around the world. In 2004, that was a fairly simple goal… and we had no idea how much our understanding...text into our work, we began to realize how the principles we used in our daily content writing applied ...writing in scientific writing, and how often the principles we developed to use in our website and educational...Steps to clear writing Let’s use Figure 1 as our example. Remember, our goal is to use words to describe...Gradient Ultracentrifugation. The reader may be, for example, a graduate student using viral vectors for the...process, try writing your own alt text for the example image. We’ll wait!  Done? Excellent! We asked our...and help make your paper accessible to even more people.  Whether you start small with alt text or immediately...

... synthetic transcription activation complex consisting of multiple distinct effector domains that work...well enough on any gene that they could be used by people with little bioengineering expertise? “This is ... Mediator or SAM. SAM is an engineered protein complex for the transcriptional activation of endogenous...with a BRAF inhibitor. (The test was the perfect complement for one conducted earlier by the Zhang lab for... These libraries are lentiviral in nature, so please ensure that you are equipped and authorized to ...sam.genome-engineering.org/. Crystal structure of Cas9 in complex with guide RNA and target DNA. Nishimasu H, Ran...transcriptional activation by an engineered CRISPR-Cas9 complex. Konermann S, Brigham MD, Trevino AE, Joung J, ...

...Senior people (of which there are never enough, especially in underrepresented groups) reach multiple mentees...share a similar direction or problem. For example, a group of people who are all interested in making a transition... at my company, but there weren't enough senior people willing to step up and be matched with the many... or both of these criteria. If you can, include people from different labs, departments or organizations... such as pre-reads, videos or exercises to be completed in preparation for the meeting or to be read or...event together and help each other practice meeting people – compete for fastest to set up a coffee date or...

...interested in expressing multiple small genes, you can design single rAAV vectors with multiple genes packaged ...indicate the ITR and serotype, respectively. For example, AAV2/8 has a type 2 ITR and a type 8 serotype....interested in heftier genes, you can coinfect with multiple viruses to express genes or gene sets over 4.7...conventional single-stranded AAV (ssAAV) and a self-complementary AAV (scAAV) vector. Full-length ssAAV vector...2.5Kb) and you need expression in a hurry, self-complementary AAV (scAAV) may be an option (McCarty et al.... E. Monahan, and Richard J. Samulski. "Self-complementary recombinant adeno-associated virus (scAAV) vectors... Multiple Authors...

...presentation, lost in a sea of data, my mind went completely blank. After several seconds of truly panicked...honest, if my paper hadn’t been ridiculously more complex than the one before it, I don’t think I would have...blocks. The problem is, when it happens to other people, it’s normal and quickly forgotten, and when it...and awful. So try to keep some perspective. Most people won't notice, or won't remember and those who do...dumpster noises were just last week!  Nerves It’s completely normal to be anxious, nervous, and/or apprehensive...still get nervous quite regularly, and I've had multiple jobs where all I did was public speaking. If you're...make a strategy that can help. Even something as simple as telling yourself, “I can stop and check my notes...

...silencing complexes (RISCs), which detect dsRNA in the cytoplasm and use it as a guide to complementary RNA ... DNA at risk. Consequences of duplexed RNA Mammalian cells have multiple receptors in place to sense dsRNA... pathogenic proteins, as well as in the lab to deplete expression of proteins of interest. RNAi has even... cytosol. Fig. 2: Endogenous sources of duplexed RNA DNA:RNA hybrids Sometimes ssRNA just needs...intermediates in the process. These transcription-coupled R-loops are transient in nature and will dissociate...dissociate when gene transcription is complete. However, transcription of thousands of genes is constantly...

...The first couples NLuc to fluorescent proteins to make better reporters and the second couples NLuc to ...are available from the repository. We encourage people to go to Addgene to get new innovative tools. After... NanoLuc® Luciferase-based tools are fantastic examples of technologies that can accelerate your research...endogenous protein tagging as it is only 19 kDa. An example is the CRISPaint-NLuc construct (Plasmid #67178...non-optimal for in vivo imaging. The solution is to couple Nluc with a red-shifted fluor. Here, NLuc internally... Nano-Lanterns  Suzuki, K., et al. (5) imaging multiple proteins at the same time in live cells using ... but has been hampered by dim signals from the coupled luciferase. This strategy was first applied using...

...QCascade complex surveys the genome and binds its target DNA sequence. The transposase complex binds the...really depends on where a transposon lands. For example, in bacteria, transposons can move antibiotic resistance...is left behind. When the transposon-transposase complex finds its target site, it integrates, which produces...TEs You can think of autonomous transposons as “complete transposons” since they encode the protein they...selecting a transposon for a mutagenesis screen. For example, piggyBac and Tol2 are best for screening promoters...DNA-targeting module with the crRNA, 3) a transposase complex made up by three transposase proteins (TnsA, TnsB...

...increases. Multiple inserts: There are some cloning methods that make working with multiple inserts a ...as some methods are more amenable to combining multiple DNA fragments than others. In today’s post, we...with a special emphasis on these five categories. Please note that many of the following cloning methodologies...interested in learning more about any one of them, please click on the links throughout the text or see the...per reaction but can be used to quickly generate multiple clones. Difficulty: If you’re an experienced cloner...Golden Gate assembly allows one to simultaneously complete an ordered set of scarless ligations following...one right answer, so don’t be afraid to explore multiple approaches to find out which one works best for...

...when studying them since their complete knockdown is lethal. Incomplete or titratable knockdown, however...the tools scientists can use to study them. For example, gene knockdown with CRISPR interference (CRISPRi...wall. Once in the recipient cell, the CRISPRi DNA (purple) flanked by left and right Tn7 end sequences (green...showed that Mobile-CRISPRi is transferable into multiple bacterial species including several involved in...pathogenic strains and environmental isolates. For example, Peters and colleagues used Mobile-CRISPRi to knockdown...if that gene is a target of an antibiotic. For example, let’s take a look at the essential gene folA which...also possible with Mobile-CRISPRi. As a proof of principle, Peters and colleagues constructed a 40-member...

...into multiple cell types of the same lineage. Blood (hematopoietic) stem cells are a good example of multipotent...mostly to maintain cell numbers or replenish dying cells. For example, hematopoietic stem cells found in...functions [1]. In many tissues, stem cells serve to replenish/replace damaged cells that no longer function ...[1]. Stem cells’ ability to differentiate into multiple cell types makes them useful models for developmental...thought of as descendants of totipotent cells. An example of pluripotent cells is embryonic stem cells (ESCs...in science communication and publishing after completing her postdoctoral training from Massachusetts ...

...yield. Keep in mind that you might need to use multiple techniques for a particularly stubborn prep. ...remove endonucleases (see figure at right for an example). Look out for carbohydrates Certain strains, including...the cell lysis process. To ensure that lysis is complete, use a reagent like Qiagen’s LyseBlue, which uses... uses color to show you if lysis is incomplete. Protein expression strains are not good for cloning Bacterial...yields. Why would volume make a difference? In the simplest of terms, more volume = more cells = more plasmid...culture media Changing to a richer media or adding supplements can improve your cell density, leading to an ...normally recommended antibiotic concentration. Add supplements to the media You can achieve higher cell density...