...some clones being outcompeted by others in liquid culture, dilute and plate the complete library of transformants...faces as it approaches a host cell surface are complex polysaccharide structures, many of which are easily... DNA from host cell nucleases (4). To further complicate matters, even when sequenced, phage genomes tend... for cell-binding abilities (6). This can be accomplished using a restriction enzyme with a short recognition...involves forcing DNA through a small opening using compressed nitrogen gas; this shears DNA at random locations...your resultant plasmid libraries into chemically competent E. coli and analyze transformants for appropriately...transformation should be performed using a highly competent strain that you can use for long-term library ...

...technology, and good computer skills will give you an advantage. Strong writing, computer skills (Word, Excel...you can fully investigate and solve a problem or complete a project, overcoming inevitable failures and ... Nature Photographer/Artist Ecologist     Computer and writing skills Writing a thesis or dissertation...which are useful to many employers. Increasingly, computer skills are highly desired, as roles are becoming...Will you be coordinating projects? Do you need computer programming skills? Other things to consider...as forensics currently have too many applicants compared to the number of available roles). Make your resume...

... interested in are compatible with your microscope optics, you will want to compare the excitation and...fluorescent proteins currently available. Choosing compatible fluorescent proteins To choose a set of fluorescent...scope of this post. Are your fluorescent proteins compatible with your microscope optics? To determine if ...is efficiently collected by the microscope. To compare the match between a fluorescent protein and a filter...here's a screenshot from the Chroma Spectra Viewer comparing a standard Cy3 or Rhodamine filter set (Chroma...channels used for more abundant proteins or marking compartments. I hope this sheds some light on multicolor ...

...Jurkat T cells Complexes with DNA; causes strand scissions 100-400 ug/mL *Not comprehensive.   ** In eukaryotes...cells. Of course, these mammalian vectors must be compatible with the cell type they are tranfected into –...cells. Before getting into the mammalian plasmid components, it may be useful to describe the means of introducing...called transfection. Transfection is somewhat comparable to bacterial transformation (the introduction...void. These ORIs, however, require additional components expressed in trans within the cell for effective...ORIs, respectively. The presence of these viral components greatly reduces the rate of plasmid dilution ...

...change. Because of this, ABEs are only weakly compatible with many Cas domains beyond SpCas9. In order...lab set out to evolve ABEs with an increased compatibility to Cas homologs. Evolving better base editors...and editing speed and consists of the following components: A plasmid in the host bacterium expressing ...phage mutation rate Phage infection results in a complete ABE protein. If the phage delivers an active variant...variants with much higher base editing activity compared to the original TadA-7.10 enzyme. After testing...which edits 590-fold faster than TadA-7.10. It is compatible with all Cas9 and Cas12 homologs that the researchers...adenine base editor with improved Cas domain compatibility and activity. Nature Biotechnology. https://...

...loss-of-function or neutral, as compared to wild type, would be extremely helpful as a complementary approach. Also, ...characterized? Some have developed exciting computational algorithms, such as SIFT or PolyPhen to approach... Genetic Pathway Reference Set. These Gateway-compatible entry clones are the product of many years of...cancer phenotypes. These assays were designed to complement the one assay per gene approach that has been...genes. These should be used to create ever more complete “look-up-tables” of mutant function that could...approach: Phenotypic Characterization of a Comprehensive Set of MAPK1/ERK2 Missense Mutants. Brenan L...

...clones for complete functional knockout of your target gene. Mismatch nuclease assays are completely “blind.... The drop-off probe is designed to perfectly complement the wild type sequence and is not capable of ...precision genome editing workflow. Digital PCR comparison to other validation methods As mentioned, these...96-well plate without prior quantification. Comparatively, mismatch assays require between 200 and 500... in a high background of wild type targets. Comparatively, detection limits of about 5% mutant targets...PMC3773023. 7. Vouillot, L., Thélie, A. & Pollet, N. Comparison of T7E1 and surveyor mismatch cleavage assays...

...this complex holds a pair of DNA ends together, forming a paired end complex The paired end complex then...then ligates compatible DNA ends together, thus repairing the break. This is a simplified, streamlined...processed. Processing occurs prior to ligation as incompatible DNA ends interfere with that step. Accordingly...pop-in” insertion) provided a template containing compatible overhangs is available. Cas9 can also be altered...Biochemistry and Biophysics and the Lineberger Comprehensive Cancer Center at UNC Chapel Hill.   References...

... Mediator or SAM. SAM is an engineered protein complex for the transcriptional activation of endogenous...endogenous genes and it consists of three components: 1. A nucleolytically inactive Cas9-VP64 fusion; 2. A sgRNA... assemble a synthetic transcription activation complex consisting of multiple distinct effector domains...with a BRAF inhibitor. (The test was the perfect complement for one conducted earlier by the Zhang lab for...gain-of-function screening when combined with a compact pooled sgRNA library. Our SAM-mediated screens ...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, ...

...becomes more of an apples-to-oranges comparison. Addgene: Can you compare the TALEN kit to CRISPR/Cas9? When...research the kit now makes possible, and how it compares to his CRISPR/Cas9 system. Addgene: For people...other cells. TALENs are engineered proteins that comprise a DNA binding domain and an endonuclease domain...especially well-suited for use in stem cells. (See the complete protocol at StemBook). Addgene: How are you now...genetic background and do an apples-to-apples comparison of all those mutations. If you make induced pluripotent...pluripotent stem (iPS) cells from patients and try to compare those lines, they may all have the mutations but...

...fantastic options for delivering shRNA, CRISPR/Cas9 components, and fluorescent sensors. Retroviral vector ...forming a vector that could create replication competent virus. Even though this system was efficient at...encoding 1) the lentiviral vector genome which was composed of the wild-type 5’ and 3’ LTRs, the ψ sequence...potential risk for the generation of replication competent lentiviruses (RCL) especially if you are working...positive cells could make the virus replication competent. The 3rd generation of lentiviral vectors was...8 kb) Potential for generation of replication-competent lentivirus (RCL) Efficient gene transfer Potential...Potential for insertional mutagenesis: Replication-incompetent lentiviruses with human tropism can still infect...

...done in reverse. Assuming you want to become competitive for a Group Leader position, what will you need...requires that all of the experiments have been completed. Therefore, to be ready to apply for a Group Leader... you get more publications, you will be more competitive for fellowships. If you explore your system more...Discussions can reveal mutual scientific interests, complementary perspectives, a shared communication style. ...job search. Therefore, you must balance these competing goals. Knowing when to follow a lead and when ...master. While the calculus of these decisions is complex, here are a few guidelines to help you evaluate... much new data, speculate on what you hope to accomplish and how your research fits in with the current...

...techniques differ in important ways, but can also complement one another by providing both broad and specific... predict off-target effects of sgRNAs using computational methods. They identify possible off-target sites...the clinic. NGS-based methods provide the most complete information profile regarding putative off-target... with in silico prediction to create a more comprehensive list of off-target editing events. Two of the...tissue from whole animal models, no exogenous component required (e.g. dsODN), doesn't require high read...Vouillot, Léna, Aurore Thélie, and Nicolas Pollet. "Comparison of T7E1 and surveyor mismatch cleavage assays...the limit of detection for Sanger sequencing: a comparison of methodologies for KRAS variant detection."...

... The Association for Creatine Deficiencies: Compensating for SLC6A8 mutations in Creatine Transporter...channels typically expressed on other cell types can compensate for or bypass the dysfunction of another solute...cells to identify any solute channels that can compensate for the defect caused by SLC6A8 deficiency. “...where one of the X chromosomes is partially or completely missing in women. Bicuspid aortic valve (an aortic...with an emphasis on oligomerization, signalling complex assembly, and localization to the nucleus,” says...at McGill University.  These protein-receptor complexes consist of many parts that work together on the...gene, which codes for G𝛃1 subunits of G protein complexes, lead to a rare type of developmental delay, and...

...minutes of inactivating the CASANOVA complex, fluorescent CRISPR complexes were observed binding telomeric ...using blue light (Bubeck et al., 2018). They accomplished this by inserting a photo-sensitive LOV2 domain...indicator podcast segment The neurons in the brain compute using electrical impulses to coordinate emotions...sensitivity, responds quickly, is photostable, and is compatible with optogenetic control. The researchers also... the “2in1” vector cloning system - a Gateway compatible approach to simultaneously clone two genes-of-interest...2015) or ratiometric Bimolecular Fluorescence Complementation (rBiFC, Grefen and Blatt, 2012). The 2in1 Plasmid...

...preserves RNA structure and naturally occurring complexes present in the cell, it can also introduce non-physiological...interest. See figure 1 below for details on the key components of RaPID. Ramanathan et al. also tweaked BirA... BirA* homolog found in B. subtilis. BASU was  compared head-to-head with the E. coli and BioID2 BirA*...streptavidin signal (~250-fold enrichment) when compared to a scrambled RNA motif control). BioID2 had ...with BASU still generated a >30-fold enrichment compared to E. coli’s BirA*. Faster labeling kinetics also...HHCS-associated sequences had lower IREB2 binding enrichment compared to wild-type sequences and that loss of binding...Russell, P., & Guttman, M. (2014). Methods for comprehensive experimental identification of RNA-protein interactions...

...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...

...cells, and animals. Some of these viruses are completely inert, but others can cause diseases. Nervous...One such control is dividing up the essential components of a virus into different plasmids. For a retroviral... are not encoded in the transfer plasmid, the component that physically integrates into the host genome...genome, the virus is replication incompetent, meaning it can’t replicate and continue the infection cycle... Figure 1: Separation of retroviral packaging components onto distinct plasmids for safety.    Viral...Viral cargo-specific safety   After replication competency, the second concern with virus safety is the ...materials, you’ll need to use aerosol-tight caps, compatible with your centrifugation speed, to cover the ...

... SCF E3 ubiquitin ligase complex. In the presence of auxin, this TIR1 complex binds to the degron sequence...depiction of chromatin within a nucleus -> DNA compaction around histones -> Examples of histone-modifying...included for visualization purposes, but the key component of the editing assemblies are sensor-actuators...The tool-kit consists of highly useful vectors compatible with Gibson and Saptrap cloning systems to generate...Resources page.  Plasmids for delivering CRISPRi components by a single AAV vector are now available from... developed by Taekjip Ha’s lab where Cas9-gRNA complex binds the target DNA without cleavage until it'...

...-Vvd showed substantially improved activity as compared to other recombinase/photodimer pairs. Created.... https://pubmed.ncbi.nlm.nih.gov/24390935/ Hypercompact genome editor CRISPR-Casɸ By Melina Fan The ...toolbox with their discovery of CRISPR-Casɸ, a hypercompact genome editor from the Biggiephage clade of ...cells. Casɸ from Biggiephage can eliminate competing mobile genetic elements. Image by Basem Al-Shayeb...the Doudna Lab. Scientists looking for more compact genome editors due to delivery constraints and ...efficiency and reduced RNA-off target footprint compared to current N-terminal linked SaCas9 ABE variants...in screens without any reduction in performance compared to larger genome-wide libraries. Find the library...

... a Cambrian-like explosion of software tools accompanying the CRISPR revolution. (See omictools for even... a 96-well plate and comes out of it. Biotech companies are known to have tools that do this internally...guides and primers in batch, optimized for HDR. Compared to wealth of resources for guide design, there...batch analysis because of its ease-of-use and comprehensive reports. With CrispyCrunch, you get the all ... ranking formula that does just this: Rank = (compressed CFD score) * (gaussian normalized cut-to-insert...is within the coding region, the algorithm then compares the guide sequence to every possible 23bp sequence...Francisco. Before starting his career in software, he completed a MSc in Psychology at McMaster University in ...

...derived from AAV9, their efficacy is generally compared to the AAV9 serotype or an engineered precursor...doesn’t lead to enhanced transgene expression, compared to AAV9, in BALB/cJ mice but is more effective..."Mouse Strain-Specific Tropism?" column. For a complete list of mice strains with LY6A-dependent tropism... by the Gradinaru lab (Deverman et al., 2016). Compared to AAV9, it effectively transduces neurons and...toward astrocytes, but its neuron transduction encompasses more molecularly-defined neuronal subtypes and...astrocytes and neurons, with greatly increased efficacy compared to AAV9 (Hanlon et al., 2019). This capsid was...brain regions, with reduced off-target expression compared to AAV9 in infant rhesus macaques. The AAV.PAL...

... stranded DNA breaks that commonly result in a complete loss of function while nuclease-deactivated Cas9...have several limitations. CRISPRko results in a complete loss of function meaning that if an essential ... more hits than the SAM library approach in a comparison study screening for vemurafenib resistance in...(Konermann et al., 2015). Root and Doench also compared the Calabrese library to screens performed with...that ORF and CRISPRa libraries can be used in complement to study gene function. Find the Calabrese pooled...gene function while using fewer sgRNAs. These new compact libraries will allow scientists to conduct genome-wide...transcriptional activation by an engineered CRISPR-Cas9 complex." Nature517.7536 (2015): 583. PubMed PMID: 25494202...

...mutations in the genome, but they’re inefficient compared to CRISPR/Cas9 edits that rely on double strand...only occur through editing by a APOBEC-Cas9n-UGI complex. So how exactly does this work? Figure 1:...editing efficiency can be easily determined by comparing cells expressing only eGFP expression to cells...DNA editing efficiency of APOBEC-Cas9 editisome complexes in real-time (St. Martin et al., 2019). To create...DNA editing efficiency of APOBEC-Cas9 editisome complexes. The eGFP reporters rely on the correction of ...frequency of genomic base pair edits of interest compared to cells segregated solely by a reporter of transfection... single base editing by APOBEC-Cas9 editosome complexes. Scientific Reports 9: . https://doi.org/10.1038...

...Construction of solid matrix-antibody-antigen complexes containing simian immunodeficiency virus p27 using...structures of CRBNmidi alone (A) and in a ternary complex with the multiple myeloma drug mezigdomide and ...on its own, with drug ligands, and in ternary complexes with other proteins, establishing CRBNmidi as ...matured considerably faster, with expression levels comparable to GFP during early embryogenesis (Figure 3).... through the middle of each embryo are shown, comparing GFP, wrmScarlet (mScarlet), and the new mScarlet3...Merritt D.M., Pe K., Cesar M., Hobert O. (2024) Comparative analysis of new mScarlet-based red fluorescent...than FRET-based mechanosensors (~60 kDa) and are compatible with common microscopy methods. Plus, the luminescence...

...neuronal function, with far fewer side effects compared to lesioning methods -  they may be the key to...camera or tracking software. Locomotion is a highly complex process on the level of individual neurons and ...different motivation (and brain areas associated) compared to an animal running to escape a threat. Locomotion...plus maze, the light-dark box is a simple device comprised of a brightly lit (risky) and a dark (protected... control group preferred staying in the dark compartment (Howerton at al. 2014). Addgene has several recombinase-expressing... passive mode, the stronger is the depressive component of the animal behavior. With the availability ...behavioral neuroscience to reveal insights into the complex relationship between genes, neurons, and behavior...

... newly formed compensation committee. The compensation committee creates a compensation philosophy that...employee compensation and also serves as an objective body to oversee some of Addgene’s compensation decisions... environment.   What do you hope Addgene will accomplish in the near future? Right now it’s a matter of...

...lengths to keep accurate records, but given the complexities of modern research, it’s often not enough. A... have software tools that help us efficiently accomplish our daily goals. These tools remind us of our...others using excel sheets or organizing folders in complex ways, there are solutions that bring more to the...-based solutions or open source solutions. In comparison to paper-based record keeping, ELNs offer functionalities... if you are working for a large institution or company, site licenses might be available as well. In most...information. Does your lab need to meet any specific compliance requirements – e.g. 21 CFR part 11, HIPAA? Answering...of time and effort and give the lab a serious competitive advantage.  Many thanks to our guest blogger...

...the N2 gene of SARS-CoV-2. If the Cas12a-sgRNA complex binds to the target sequences, Cas12a activity ... amplified region of interest. Cas13 uses a complementary crRNA sequence to bind to the sample and upon...steps and the Cas protein used. Here, all the components needed for the reaction are mixed in a single...incubated at one temperature. Two Cas12a-crRNA complexes are designed, each binding to one strand of the... RPA primers, the Cas12a-crRNA binds to the complementary region in the target sequence and this activity... substrates for Cas13. Cas13, upon finding complementarity between the gRNA and a given fluorescently ...streptavidin coated beads. Fluorescently labelled dCas9 complexes containing sgRNA that have no mismatches bind ...

...websites compiling antibody data that can help you compare antibodies from different companies, such as... a fluorophore (Figure 1).   Figure 1: Comparison between how direct immunofluorescence and indirect....  How do you find validated antibodies? Many companies that produce and distribute antibodies validate...through their publications or as a review on a company website. It is important to review the data and... a couple of antibodies that seem promising to compare in your own lab. Even with all of the resources...

...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...

...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 ...

...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...

... 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...

...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...