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

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

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

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

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

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

...years since its discovery, researchers have done significant work to improve the Adv genome’s utility in research... quickly due to immune response against AdV. Importantly, recombination-competent adenovirus (RCA) can...formation assay on A549 cells . Alternatively, antibody-based assays are commercially available for faster...more complex and the presence of helper virus contaminants have slowed the use of this system. New and ...detailed instructions on how to generate the final recombinant adenoviral plasmid construct, please see Addgene... references 1, 2.*Pro-Tip*: once the correct recombinant pAdV plasmid is identified, re-transform into...2-3 weeks) - Here you’ll produce the primary recombinant adenoviral stock (rAdV-S). HEK293 cells are transfected...

...degradation of caffeine (1,3,7-trimethylxanthine) and its conversion into xanthine. The majority of this operon...enzyme required for conversion of xanthine into guanine, and without xanthine supplied in the media, these ...desired due to its very low quantum efficiency. ShadowY has a lower quantum yield and higher absorption...material for the bioproduction of therapeutic methylxanthine drugs like theophylline. To facilitate future... glutathione S-transferase homolog from a Janthinobacterium sp. Moreover, a caffeine “addicted” E. coli... of this knockout strain can be rescued by the xanthine produced via complete demethylation of caffeine... assays. ShadowG is darker than other dark YFP mutants, it reduces the risk of spectral contamination ...

...seriously hinder gene therapy outcomes. Anti-AAV antibodies developed by the immune system – patients...To date, gene therapy has been used to treat 10 infants with this disease. To do so, their T-cells were...and the T-cells were transferred back into the infants. Almost all patients have achieved persistent immunological...immunogenic profile. AAV was first discovered as a contaminant of adenovirus preparations. It is an icosahedral...disease, and HIV. A notable landmark study showed significant clinical efficacy against a form of blindness... of LCA and mutations in RPE65 (n=3), showed significant improvements in vision over a period of 1 to ...infection. For example, prevalence of neutralizing antibodies against some AAV types, AAV1, 2, 3 and 5 are ...

...to solve, we want to focus our research on worm locomotion, but our worms are constantly moving so we ...complexity using existing C. elegans mutants. For instance, we use a mutant known as CX51 that exhibits motor...real mutant worm. Like many academic labs, we have the capability to study many different mutants because...educational institution meaning that we can obtain known mutants at $7.00 a strain. Makerspaces like ours can also...model  so that we can use CRISPR/cas9 to build the mutants, analyse their motor behaviour, and ultimately ...month - with only occasional supplementation with grant funds, money is always tight. Of course, unlike ...monitoring homemade microfluidic chips using the fantastic optics and fluidics platform it contains. Biotech...

...transform your resultant plasmid libraries into chemically competent E. coli and analyze transformants for appropriately...characterization, these proteins could function as surrogate antibodies for identifying and characterizing sugars and...Essentially, any method that results in the bulk of resultant fragments falling between 1 and 3 kb results in...culture, dilute and plate the complete library of transformants for single colonies (this usually requires 10...DNA directly from the colonies. Transform the resultant plasmid DNA into E. coli BL21 (DE3) cells (these... protein production), then dilute and plate transformants for isolated colonies (150-200 per plate) across...then be exposed to a lysis buffer to release recombinant proteins directly onto the nitrocellulose for...

... used to identify antibiotic resistance genes. Genes that are targets of antibiotics are often essential...for CRISPRi transfer Mobile-CRISPRi offers two advantages over existing bacterial CRISPRi methods. First...customization with strain-specific promoters, antibiotic resistance selection markers, gRNAs, and dCas9...downstream of the glmS gene in the recipient's genome. Antibiotic plates lacking DAP select against donor bacteria...The ICE integrates into the trnS-leu2 locus. Antibiotics select for recipient bacteria with an integrated...experiments where maintaining library diversity is important, while lower transfer rates are only suited for...researchers to study if that gene is a target of an antibiotic. For example, let’s take a look at the essential...

...to try developing a variant with an enhanced response. Evolution of a new variant Maria and the team set...project, Maria’s screens of AsLOV2 variants showed some significant gains! After a few more rounds of ...mutagenesis and screening, the EGFP and mScarlet variants were showing no obvious signs of improved response...the magnetic field (Figure 2); they named this variant MagLOV. Andrew was thrilled to see that MagLOV’...: Fluorescence intensity and ΔF/F of MagLOV, a variant of AsLOV2 with mutations C450P, L496V, Q513K, G528K... Andrew and Maria expect that further-improved variants of MagLOV will be possible with other engineering...other researchers from a range of fields, like quantum chemistry and protein biophysics, as well as inventors...

...noncovalent dimers. This property of EGFP can lead to significant artifacts. If you're using green fluorescent ..., and behavior of a POI. As an investigator, you want a fusion tag to be inert, to not produce artifacts...making FPs monomeric, yet many investigators remain ignorant of FP dimerization. Equally problematic, several...its molecular affinity, termed the dissociation constant or Kd, and its concentration. The smaller the ...volume. In many biological research situations, the relevant volume is that of the cell. For the physical behaviors... Fusion of an FP to a transmembrane protein significantly increases the effective concentration of the...biosensors were made with CFP and YFP, cyan and yellow variants of EGFP equally capable of dimerizing as EGFP....

...example we’re going to be looking for recombinant monoclonal antibody plasmids from the James Trimmer Lab...better. Here, I wanted to find a specific plasmid from the NeuroMab collection for an antibody against PARIS... terms used, so you will only be shown filters relevant to your current search. We’ll take a closer look...look at filters in a moment. First, I want to highlight the convenient ways our new search tool lets you...distributing 94 plasmids expressing validated antibodies for neuroscience research (see the NeuroMab Collection...plasmid that would be useful in an experiment, and want to see if someone has already created and deposited...and filters change as we now only show options relevant to your current search terms and potential results...

...either transcript variant led to robust and specific upregulation of the targeted variants without inducing...activation? To summarize, CRISPRa uses a nuclease-dead variant of Cas9 (dCas9) and engineered combinations of ...Figure 1a for the transgenic construct). More importantly, the expression of this construct is Cre-dependent...used their optimized vectors to highlight a key advantage of CRISPRa over traditional cDNA overexpression... gene isoform individually. To highlight this advantage, they selected the immediate-early gene Bdnf, ...inducing expression of the non-targeted variant. Moreover, isoform-specific expression of Bdnf resulted in... between the Bdnf I and Bdnf IV overexpression variants were other immediate-early genes associated with...

...genes important for mesoderm differentiation Now, let's turn tinman and bagpipe, two genes important for...protein Groucho, a transcriptional co-repressor important in development. Groucho family proteins are found... in neural development, sex determination, and anterior-posterior segmentation (Jennings and Ish-Horowicz...Dana Farber Cancer Institute! Sonic Hedgehog, an important regulator of embryonic development The largest...video game character but rather to an extremely important gene in animal development. To understand where...name but some of our Addgenies had an idea: sqh mutant fly ovaries had “squiggly” actin fibers that resemble...spermatocytes to enter meiosis. Testis of cookie monster mutant flies are filled with early mitotic cells as well...

...aptamers hold a few important advantages over their amino acid-based counterparts, antibodies and enzymes, such...applications, including detection of environmental pollutants, drug discovery, and disease diagnosis. As biosensors...commercial synthesis (those of us who have used antibodies for diagnostics know how frustrating it can be...hybridization (FISH) where fluorophore-linked antibodies are used to visualize RNA. Low signal-to-noise...only in this case ligand binding would bring the relevant ends together rather than separate them (Fig. ...quenchers are still relatively bulky. One of the advantages of oligonucleotides over proteins is their modularity... Years later, Samie Jaffrey’s laboratory took advantage of their deep knowledge of the nature of GFP fluorescence...

...expression mRNA Plant promoter from maize ubiquitin gene Constitutive  Gives high expression in plants. H1 small...look at some of its parts. Elements such as the antibiotic resistance gene and the origin of replication...with the type of RNA that needs to be made: if you want mRNA (for gene expression) you need to use an RNAP...various eukaryotic cell types (mammalian, yeast, plants, etc) require unique promoters and there is very...expression. CaMV35S General expression mRNA Strong plant promoter from the Cauliflower Mosaic Virus Constitutive...transcription, the promoter drives either the sense OR antisense transcript depending on its orientation to your...

... released a new variant of jGCaMP7 (keep reading) and will be releasing more variants in the next few ...few months (see “coming soon”). The 7b variant (pGP-AAV-syn-FLEX-jGCaMP7b-WPRE) exhibits the brightest resting...processes like dendrites and axons. Find the 7b variant of jGCaMP7 in AAV1 at Addgene! See all calcium ...membrane localization and brightness. The F102G variant (pAAV.hSyn-FLEX.iGABASnFR.F102G) is noticeably ...responses to GABA. Find the iGABASnFR and its F102G variant in AAV1 at Addgene! Archon1, a voltage sensor AAV...electrical activity in the brain was measured with implanted electrodes. The Archon1 voltage reporter allows... vectors can be used to visualize cells and to quantify targeting of particular cell types. For example...