We narrowed to 614 results for: cat.1

...available from their original sources. Figure 1: Biological repositories that distribute mouse strains... from Reproducibility for Everyone. Case study 1: Cell lines Many of the reagents used to investigate...COVID-19: an observational study. The Lancet Microbe 1:e14–e23 . https://doi.org/10.1016/s2666-5247(20)30004...tropism, replication kinetics, and cell damage profiling of SARS-CoV-2 and SARS-CoV with implications for ... Another example of reagents that have broad applications but have proven useful to the study of SARS-...vectors VSV-G and psPAX2, which were used in a publication by Xiuyuan Ou, et al. that characterized the ... requested these plasmids from Addgene. This publication then resulted in the development of a new plasmid...

...s pLKO.1 protocol. Pro-tip: this plasmid grows more slowly than standard plasmids. Find pLKO.1 - TRC cloning...experiments.  Find the scrambled shRNA vector. pLKO.1 - TRC cloning vector - This vector from the David ...taken us on many adventures...From moving office locations three times, to opening our UK office in 2014,...cell types so it is useful in a wide range of applications. This plasmid was the top requested plasmid ...notice the “V2.0” at the end of this plasmid which indicates that there was a prior version of this plasmid...plasmid was called pLKO-Tet-On in the original publication but its name was subsequently changed to clarify...

...available. Table 1 compares some general advantages and disadvantages of each approach. Table 1: Properties...alternatives to fluorescent proteins. Figure 1: Firefly, from Wikimedia Commons. The North American...American firefly, Photinus pyralis, catalyzes a chemical reaction in its abdomen to produce yellow-green... Figure 2: Simplified chemical reaction catalyzed by luciferase, releasing light. Created with BioRender.com...NanoBiT, are expressed in two subunits and only catalyze a luminescent reaction when they come together...

...digital) signal at the end of the reaction (Figure 1). Partitions that did not contain any target DNA at...integrated through homology-directed repair (HDR) (1), or indel mutations resulting from non-homologous...Follow him on Twitter @AnotherLabRat.     References 1. Miyaoka, Y. et al. Isolation of single-base genome-edited...a gene of interest is one of the most common applications of precision genome editing, the remainder of...single molecule resolution of digital PCR allows classification of individual target molecules based on their...single wells of a 96-well plate without prior quantification. Comparatively, mismatch assays require between...

...standard for genetically-encoded FRET donors (Fig. 1). Scores of FP variants have been created, thereby...with finesse using tRNA suppression technology (Fig. 1) (Dumas, Lercher, Spicer, & Davis, 2015). By using...orthogonal labeling strategies are presented (Fig. 1) which can be carried out using Addgene plasmids. ...involved in skeletal muscle contraction.   References 1. Degorce, François, et al. "HTRF: a technology tailored... transfer measurements of ryanodine receptor type 1 structure using a novel site-specific labeling method...cellular impediments. FRET has been used in many applications, including drug discovery by big pharma (i.e... acceptor doesn’t need to go to the same exact location as the donor. As long as some of the acceptor ...

...and CasPEDIA have got you covered!     Fig. 1 – CasPEDIA Cas ID display information for a given ...established uses, and intended applications. Within CasPEDIA, there is a classification system, CasID, to help...multiplex ability Experimental guidelines and applications Protein structure and properties Links to ...you want to read literature on the less common applications of an enzyme for your non-traditional experiment...al. (2023). CasPEDIA Database: A Functional Classification System for Class 2 CRISPR-Cas Enzymes [Preprint...I., Iranzo, J., et al. (2019). Evolutionary classification of CRISPR–Cas systems: a burst of class 2 and...

...visualizing hM4D(Gi)-mCherry in fixed tissue. References 1. Aschauer DF, Kreuz S, Rumpel S. Analysis of transduction...efficiency, tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain. PLoS One. 2013...PMC3069789. 3. Kügler S, Kilic E, Bähr M. Human synapsin 1 gene promoter confers highly neuron-specific long-... preps. Cas9 is the headliner of most CRISPR applications and can be expressed from your cell line of ...Find Ready-to-Use Lentivirus Find Ready-to-Use AAV Catch up on Chemogenetics ...

... you’d like to use to generate your new fly line: 1. Where would you like the gene to be incorporated ...control exactly in which locus your transgene ends up (1): Random insertion has the advantage that you can...he still reads “#” as a “pound symbol”. References 1. J. R. Bateman, et al. Site-Specific Transformation...beforehand on which chromosome your docking site is located). This amounts to a small difference of two or ...throughout the lifespan of the fly to determine the implications of your genetic manipulations. You can produce...

...) vector (Figure 1). Everything else outside the ITRs gets left behind.    Figure 1: Recombinant AAV ...productivity but increases transgene expression. Sci Rep, 7(1), 5432. https://doi.org/10.1038/s41598-017-04054-4... ITRs serve as the origin of replication and are required for replication, packaging, and vector persistence...viral rep (replication) and cap (capsid) proteins, which bind to the ITRs and initiate replication. ITRs are...structures, ITRs are prone to replication errors and disruptive mutations and truncations that can make vector ...binding site for Rep78 and Rep68 to initiate replication and a terminal resolution site that serves as...are noted. Created with biorender.com. ITR modifications and types  Since ITRs are one of the most important...

...genetically targetable fluorescent indicators. Nature protocols. 2006 Aug;1(3):1057-65. Resources on Addgene.org...want a low affinity calcium biosensor, so that the (1) binding of calcium, (2) allosteric rearrangements... to a red fluorescent state only by simultaneous (1) user-controlled illumination with violet light and... a calcium sensor Genetically encoded calcium indicators (GECIs) are fluorescent proteins linked to calcium...in living cells and organisms. Unlike chemical indicators, these DNA constructs can be delivered to intact...the sensors discussed below fall into this GECI category.   For calcium sensors, one size doesn...:509-18. Inoue M. Genetically encoded calcium indicators to probe complex brain circuit dynamics in vivo...

...effective recovery of knock-ins, but did result in 1-2 kilobases of additional sequence being introduced...facilitating recovery of edited animals. Figure 1: In the background are 504 restriction digests of ...selection for repair of a temperature-sensitive pha-1 mutation significantly enriches for knock-in of 2x... 3xFLAG epitopes into other, non-linked loci; pha-1(e2123) mutant worms are perfectly viable at 15 ºC,... repair pathway. Reagents required to perform pha-1 co-conversion are available through Addgene. In 2019...Mello lab also reported that modifying the 5’ modification of oligos used to generate linear dsDNA repair...Efficient Marker-Free Recovery of Custom Genetic Modifications with CRISPR/Cas9 in Caenorhabditis elegans. ...

...difficult-to-manipulate primary cells, achieving knockdown within 1-2 hr of reagent delivery. TRIM-Away is suitable for... inducer levels. That is, if transcription factor 1 controls expression of transcription factor 2, which...modulating the induction of transcription factors 1 and 2. See Aranda-Díaz et al. for more information...changes in expression of TRIM21-associated genes, indicating that TRIM21 overexpression does not aberrantly...light microscopy, comes from his recent Nature Communications article “SRpHi ratiometric pH biosensors for...250 nm), and studying endocytosis is further complicated by the reliance on pH-based biosensors used to...the Hell lab designed their super-resolution pH indicators (SRpHi) biosensors to meet all of them. These...

...Collection Fluorescent tagging strategy Figure 1: Gene editing is the addition of a useful coding sequence...plasmid design strategy. A donor plasmid contains 1 kb homology arms matching the gene of interest, a ...Institute website at alleninstitute.org. References 1. Roberts, Brock, et al. "Systematic gene tagging using...visit the Cell Catalog on the Allen Cell Explorer, where you can also find the certificate of analysis provided...signal that makes it hard to visualize the precise location of the protein or structure of interest. Endogenous...technology, inserting large tags into a precise location in the genome is still inefficient, particularly...

...domain (Figure 1).  Figure 1: Examples of sites on antibodies used for conjugation. 1) Endogenous ...These properties allow for efficient signal amplification, making biotin conjugated antibodies good options...especially if you start to consider clinical applications). But there are a few other classes that we ...proximity ligation, immuno-PCR, and single-cell applications (Hegazy, et al., 2020; Niemeyer, et al., 2007...also conjugate antibodies directly to beads for purification purposes.  Coupling up - antibody conjugation...added to antibodies post-translationally. 4) Modifications made to antibodies, such as inclusion of non-canonical...Detecting Protein-Protein Interactions and Protein Modifications in Cells and Tissues in Situ. Curr Protoc Cell...

...for particular applications. Read on to learn more about each reporter or check out table 1 for a summary...with a standard primary/secondary antibody. Table 1: Summary of the Key Features of HRP, Spaghetti Monster...NIH’s Developmental Studies Hybridoma Bank (DSHB). 1 ml of antibody-containing hybridoma supernatant is...detected with tyramide signal amplification. Tyramide signal amplification occurs when HRP converts a fluorescently...reporters (i.e. fluorescent dyes, HRP, etc. ) after purification. Antibodies are difficult for an individual ...culture media. Their His tags allow for further purification, if desired, but it's not required for their...Pros Cons Antigens HRP (P) Tyramide signal amplification of HRP Staining cultured cells and tissue sections...

...Dronpa’s color thus indicates MEK1’s current state (Green when off, dark when on). Figure 1: Photoswitchable...few examples demonstrated in Zhou et al. 1. Testing kinase inhibitors Zhou et al examined the ability ...the protein and pdDronpa are preserved. References 1. Zhou, Xin X., Linlin Z. Fan, Pengpeng Li, Kang Shen...members of their pathways, but where the kinases are located, their signal strength, and how long their signals...domains were inserted at sites homologous to the locations they were attached to in MEK. The photoswitchable...kinases good for? There are a number of potential applications for photoswitchable kinases.  Below are a few...signaling by using psMEK1 and an ERK kinase translocation reporter with a mRuby2 fluorescent reporter ...

... in Fig. 1). Of course, this was problematic, because the DREADD receptors need to be located on the plasma... in both species (bottom panel in Fig. 1). Our results indicate that the tag protein may have an important...protein of interest the membrane.   Figure 1: Electron microscope images showing ultrastructural...red fluorescent proteins) allow simultaneous identification of more than one protein of interest. However...fluorescence expressed in the neuropil conceals identification of opsin-expressing cells). For other things...Rodgers L, Wilson IA, Lerner RA, Wigler M (1988) Purification of a RAS-responsive adenylyl cyclase complex...(2013) Overview of Affinity Tags for Protein Purification. Current Protocols in Protein Science 73: . ...

...separation is maintained by 2 sets of interactions : 1. Interactions between the non-target strand and a ...theoretically decrease off-target cutting. Figure 1: Strand separation and target DNA binding to the Cas9... improvements in genome engineering! References 1. Slaymaker, Ian M., et al. "Rationally engineered ...As evidenced by all the CRISPR publications, press, and plasmids out there, it’s obvious that CRISPR ...nickase variant, lowering Cas9 expression, and truncating gRNA sequences used for targeting; however, these...on-target and off-target activity at multiple genomic locations in HEK cells. Further testing of two of the mutants...PubMed PMID: 26628643. Find plasmids from this publication at Addgene. 2. Nishimasu, Hiroshi, et al. "Crystal...

... of the biotin-avidin interaction   Figure 1: Biotin is enriched in egg yolk while its binding ... substrate (Holden and Cremer, 2003). References 1. Chapman-Smith, A., & Cronan, J. E. “Molecular Biology...matrices.” Journal of Nanobiotechnology 13.1 (2015): 1–14. PubMed PMID: 25980573. PubMed Central PMCID: PMC4461904...detection by northern blotting.” Rice 7.1 (2014): 1–7. PubMed PMID: 26224555. PubMed Central PMCID: PMC4884002...molecular biology applications for biotin and avidin. A short list of some other common applications and links...downstream application. Biotin labelled oligonucleotides can be used in a multitude of applications including... carboxylase and decarboxylases. These enzymes catalyze the transfer of carboxyl groups to organic acids...

... requires selection. These two steps are (Figure 1): The initial plasmid integration step (the so-called...antibiotic resistance marker from the vector (abR, Figure 1). The resolution step (“double crossover”) which leads... loss of a specific phenotype (using sacB, Figure 1, further discussed below).  Counter-selectable marker...Current protocols in molecular biology 106.1 (2014): 1-16. PubMed PMID: 24733238. Additional resources on...resistance cassette, and the R6K origin of replication to allow replication only in hosts carrying the bacteriophage...chromosome. These methods roughly fall into two categories: those that rely on exogenous enzymes and those...Lambda Red recombineering, which uses enzymes domesticated from the Lambda bacteriophage, is arguably the...

...Principles and Clinical Application. Clinical and Developmental Immunology 2012:1–15 . https://doi.org/10.1155...the other (Sandomenico et al., 2020). Figure 1: Comparison between the IgG antibody and scFv. ...intensity of a tagged protein. SunTag has two components: 1) a protein of interest that’s tagged with 10-24 copies...without disulfide bonds, made by molecular evolution 1 1Edited by I. A. Wilson. Journal of Molecular Biology...led to their application for basic and translational research.  Basic research applications of scFvs: ...blood clearance, which is useful for imaging applications; better tissue penetration which is useful for... for therapeutic and imaging applications; and reduced immunogenicity when administered in vivo due to...

...mutation site, indicating that repair proceeded through NHEJ rather than HDR. Figure 1: Homology-directed... employing HDR. Ma et al. moved forward with gRNA 1 to conduct microinjection experiments in S phase zygotes...instead from the mother’s wild type allele (Figure 1). 72.4% of embryos carried two wild-type copies of...thus allowing repair to take place before DNA replication. Indeed, they observed that 41/42 embryos were...attributable to CRISPR editing. Conclusions & implications One striking feature of this paper is the failure... need to be much, much lower before clinical applications could be considered. It is important to consider...experiments are needed before we can consider clinical applications for germline editing. Although this paper represents...

...glassware should be treated with a DEPC solution for 1 hour, and autoclaved to remove residual DEPC. Alternatively...more large, expensive ultracentrifuge.  Figure 1: An outline of the different steps in RNA extraction...extraction: twenty-something years on. Nature Protocols 1:581–585. https://doi.org/10.1038/nprot.2006.83 Additional...of DNA that can potentially complicate results of a downstream application such as quantitative PCR to...easier. Recently, we published a blog on DNA purification without a kit that outlined several reasons ...take care whenever doing any type of DNA or RNA purification to avoid contamination, take extra care when..., that isn’t the case for RNA extraction and purification. There is one simple method that works, and ...

...various cyan fluorescent proteins is depicted in figure 1. Of note, the photobleaching rate does not depend ...to be used with the fluorescent protein. In figure 1 we provide an example of such a comparison between...the variants is comparable (lower panel of figure 1). To avoid changes in excitation power, it is important... localized fusion proteins and do not require dedicated plasmids or constructs. It should be noted that...reached at high power may not translate to real applications in which far less power is used. For example...insight into the photostability in the intended application (Goedhart et al, 2012). The third issue relates...Selecting a photostable protein The issues with quantification of photobleaching rates can be summarized as...

...succumb to the toxicity of CcdB.   Figure 1: Image from Bernard, P., et al. showing the pKIL 18...submit a guest blog entry of your own! References 1. Bernard, P. “Positive Selection of Recombinant DNA... plasmid ccdB killer gene”. Gene. 1994 Oct 11;148(1):71-4. PubMed PMID: 7926841. 4. Bernard, P., et al...time-consuming aspects of traditional cloning is the identification of clones that actually contain your insert...technology. CcdB: A potent toxin... The ccdB gene, located on the F sex factor plasmid of E. coli, is part...enzyme-mediated disruption of ccdB, leading to positive identification of desired clones.   ...Becomes a powerful...which can then be used for downstream cloning applications. While these two strains ultimately perform ...

...using the format indicated below and pool-clone them into the pAWp28 storage vector (Figure 1): Forward oligo..., including those found at Addgene! Figure 1: Strategy for assembling the barcoded gRNA library... He can be contacted at [email protected].   References 1. Cheng, Allen A., Huiming Ding, and Timothy K. Lu....realization of a variety of perturbations and applications in functional genomics, cell reprogramming, ...CRISPR/Optogentics Tools Resources on Addgene.org Catch up on Your CRISPR Background with Our CRISPR Guide...

...Transfer Vectors   Expressing your gene of interest pMKO.1 GFP (Plasmid #10676) Retroviral vector for cloning...GFP expression.  Cloning and expressing RNAi pMKO.1 puro (Plasmid #8452) RNA interference (RNAi) is an...information and a detailed protocol on how to use the pLKO.1 vector can be found here. Cre-Lox technology Puro....-humanized (Plasmid #44246) Expression of a catalytically inactive (dead), human codon-optimized Cas9 ...be used to construct complex lentiviruses for modification of mammalian cells with a single viral infection...Gammaretroviral Vectors: Biology, Technology and Application. Viruses. 2011. PubMed PMID: 21994751. PubMed...

... experimented with a group mentoring format where 1 mentor met with a group of mentees to get more “bang...The groups in these programs typically consist of 1-2 mentors and 3-5 mentees who meet together for an...get together. If you all work at the same place, a 1-2 hour lunch on a regular schedule might work. If ...This is a chance to practice leadership and communication skills. The leader also provides materials such...

...neuron which generates an action potential (Figure 1). This light-induced excitation can take place in ...channelrhodopsin activation to a small volume of brain tissue (~1 mm3). Current channelrhodopsins also need high-intensity...fiber-optic cables into the brain. Figure 1: With no blue light, the channelrhodopsin remains ...tegmental area (VTA) of the brain. Since the VTA is located in the midbrain, fiber-optic cannulas were still...right secondary motor cortex (M2). Since M2 is located on the surface of the brain, to deliver light to...

...point mutations with unc- 119 + selection." MicroPublication 1 (2018): 2–3. Wang, Han, et al. "cGAL, a temperature-robust...Zhang et al., 2015). In this system a modified TIR-1 protein can degrade degron-tagged proteins in an auxin...splitting cGAL4 in two and binding each half to a gp41-1-N-intein. Split inteins are protein domains that naturally...the OpenWorm website. microPublication microPublication is a unique publication for the worm community...into larger publications and would otherwise be lost in lab notebooks forever. microPublications are peer-reviewed...aims and approach of CeNGEN, check out their publication.   3’UTRome The 3’UTRs of mRNA are important ...normally one figure and can be negative and/or even replicated studies to show reproducible results. This a ...

...Recently I learned that Addgene’s pLKO.1 cloning protocol is viewed around 3,000 times each month. I ...on the Addgene website in December 2006, the pLKO.1-TRC cloning vector (deposited by David Root of the...would the thousands of other scientists using pLKO.1 variants learn about those updates? This is a problem...manuscript to get reviewed and edited prior to publication, and this will delay the dissemination of your...deposited by George Church’s lab). Addgene has also cataloged a variety of useful molecular cloning protocols...bacterial transformation. And more plasmid-related educational resources can be found in our Plasmids 101 blog...bring your phone into the lab? Maybe having a dedicated lab-use tablet? The software also makes it easy...

...nucleotides are represented equally, that’s only a 1/16 chance of having a PAM at a given site. While it... NYN/NRN NYN is less effective than NRN Table 1 - Summary of PAM-flexible Cas enzymes Are we truly...editable with CRISPR technology.     Fig. 1: SpCas9 has sparse PAM coverage in the human genome...flexibility in the location of the cut and subsequent edit. In these applications, location takes priority...make a choice about what is your top priority: location or efficiency. If you are just trying to shred...still leave you choosing between efficiency and location.   Nearly PAM-less editing   The Kleinstiver lab...in double stranded cleavage and base editing applications, with high editing capacity in both contexts...

...targeted expression. Surprisingly, <1% of synthetic promoters replicated the expression specificity of the...used four different design strategies: Strategy #1 (ProA): Create promoters with sequences upstream of...active in retinal cell types in mice. Figure 1: The four strategies used to design synthetic promoters...channelrhodopsin, fused to a fluorescent GFP marker (CatCH-GFP). The lab deposited AAV plasmids containing ...reproducibly labeled retinal cells can be found here.  Applications for synthetic promoter AAVs Besides restricting...synthetic promoters have a few other potential applications: Using promoter combinations for targeted expression... promoters expressed in. The lab tested this application using one promoter to drive expression of Cre-mCherry...

..., check out Addgene's CRISPR Guide. Figure 1: Like this jar of jellybeans, a pooled CRISPR library... selecting a CRISPR library for your experiments. 1.  What species are your cells derived from? Currently...a different gene. Libraries may be available in a 1-plasmid system, in which Cas9 is included on the gRNA-containing... unique compared to the rest of the genome and located immediately 5’ to a protospacer adjacent motif ...coli, and T. gondii genes.  2) What genetic modification are you trying to make? Addgene carries CRISPR...next-generation sequencing to check the quality of your amplification. If you've obtained a ready-to-use lentiviral... As such, sequencing CRISPR libraries after amplification and after a screen requires the use of next-...

...backbones for shRNA expression, such as pLKO.1 puro and pLKO.1 – TRC cloning vector. Lentiviral Plasmids ...they were added to the repository, including beta-catenin reporter M50 Super 8x TOPFlash, scramble shRNA ... GCaMP6 series of genetically encoded calcium indicators were the hot technology of the year in our collection...

...unlimited supply of identical antibodies (See Fig 1.). rAbs are a relatively new technology that takes...conveniently modified and engineered.   Fig. 1: Antibody production methods    NeuroMab and others... the signal. If the exposure time is long (e.g. > 1/7s) then the signal is weak and vice versa for a strong...the best, or even which one will work for your application? Like many scientists, I have spent hours sifting...produced from a new set of animals), and wrong application (different experiments/conditions can change ...differently over time), compatible with the desired applications (such as immunohistochemistry (IHC) or western...colleagues! Dr. Wu and his lab members (like me!) are dedicated to creating a public and accessible database for...

...-public settings. Ask over coffee.  Have regular 1:1 meetings with team members (don’t become known for...Scientists series will focus on an aspect of communication especially important for a manager – getting... Creating a culture of excellent, effective communication between all members of a team. As described ... opinions in meetings, via email, at formal communications events or at informal gatherings, like at lunch...

...about HIV cure research and science communication.     References 1. Gaspar, H. B., S. Howe, and A. J. ...reconstitution with a normally functioning T cell repertoire (1). In this example, it was possible to transduce the...significant improvements in vision over a period of 1 to 3 years – the treatment appears to be both safe...affinity-purified receptor-targeted viral vectors." Nature communications 6 (2015): ncomms7246. PubMed PMID: 25665714...our AAV Production Protocol Check out our AAV Purification Protocol ...

...and accessible education in gene editing and synthetic biology. Nature Communications, 15(1), 6563. https...more.  Figure 1: Workflow for cell-surface protein labeling and identification. Reproduced from Wu...with a membrane-impermeable biotin substrate (Figure 1). Biotinylated proteins were isolated, identified ...564–571. https://doi.org/10.1038/s41586-022-05682-1 Zhang, Y., et al. (2025). Probing condensate microenvironments...cell mitochondrial transfer tracker Red calcium indicators PinkyCaMP and FRCaMPi Micropeptide killswitch...the Cre-tagged mitochondria, Cre is released, translocates to the nuclease, and excises the DsRed-STOP ...doi.org/10.1073/pnas.0510511103      Red calcium indicators PinkyCaMP and FRCaMPi By Mike Lacy Many popular...

...depend on your intended application and available resources.     Figure 1: Table summarizing the... a good option for applications like immunohistochemistry on human tissues, when (1) other strategies ...expected in another. You’ll need to validate that points 1 and 2 hold true under your specific experimental conditions...validation: A view from the mountains. New Biotechnol 45:1–8. https://doi.org/10.1016/j.nbt.2018.08.002  Uhlen...intended application - Remember, just because your antibody performs as expected in one application does ...strategies for antibody validation in different applications. Here, I want to highlight the general suggestions...intentionally generalizable and not specific to certain applications or antibody targets. That said, not every strategy...

...Bacillus. Appl Microbiol Biotechnol. 2019 Jan;103(1):303-313. doi: 10.1007/s00253-018-9485-4. Epub 2018...and clonal analysis in Drosophila.” Nat Methods 9(1) (2011): 47-55. PubMed PMID: 22205518. PubMed Central...Methods Mol Biol. 2016;1478:53-78. doi: 10.1007/978-1-4939-6371-3_3. PMID: 27730575; PMCID: PMC5270762. ... and lac promoters.” Proc Natl Acad Sci U S A. 80(1) (1983): 21-5. PubMed PMID: 6337371. PubMed Central... also been developed which don’t fit in either category, but those are a subject for a future post! ... GAL80 under different promoters and create sophisticated patterns of UAS-driven gene expression. To further... PMCID: PMC2942869. Find plasmids from this publication at Addgene. del Valle Rodriguez, Alberto, Dominic...

...School Advice Part 1: Picking a Lab and a Project That’s it for now. As I indicated in the opening paragraph... products). I picked this post for a few reasons: 1) I think phage are super cool and have considered ...Plasmids at a Low Price: Kits from Addgene Savvy Advocates Needed to Navigate a Scientific Enterprise in ...

...initiating cleavage of the transcript.   Figure 1: A predicted conserved secondary structure and sequence...prokaryotic terminators and deposited several with Addgene (1). Prokaryotic polyadenylation Although mostly thought...constitutive RNA transport element lead to enhanced HIV-1 Gag expression in a context-dependent manner. Nucleic...type are generally broken down into two broad categories, prokaryotic or eukaryotic, based on the functional...termination mechanisms fall under two general categories: rho-dependent and rho-independent. Rho factor...

...expression. Brown et al., Genesis 2014 Jan;52(1):39-48. APEX2 for proteomic mapping and electron...engineering cells that lack release factor one (RF-1; Bacterial strain EcAR7) and minimizing the set of... of human mitogen-activated ERK activating kinase 1 (MEK1) with either one or two phosphoserine residues...comprised of three sets of cloning vectors (Level 0, 1, or 2) which can be utilized in three successive assembly... vectors are directionally assembled into a Level 1 vector creating a single transcriptional unit (Ex:...coding region, and terminator). Next, up to six Level 1 modules can be similarly assembled into a Level 2 ...vectors to allow for additional iterations of Level 1 assembly if necessary. Combining multiple Level 2 ...

...” Nat Protoc. 11(1): 118-33. PMID: 26678082. Find the plasmids from this publication at Addgene. 2. ...homologies. Abbreviated protocol for PITCh Step 1: Generate microhomologies in the PITCh vector ~20...editing possibilities MMEJ can enable. References 1. Sakuma, Tatsushi, et al. (2016).  “MMEJ-assisted gene...another method for making targeted modifications. A recent publication by Zhang et al. shows just that -...about a month and a half, without the need for complicated cloning of homology arms. MMEJ: An Introduction...required by MMEJ can easily be added via PCR amplification. Given the inefficiency of HDR for knock-ins... and 3’ junctions, respectively. These results indicate that PITCh is a robust method for genomic insertion...

...some technical obstacles when working with virus: 1. Viral vectors undergo DNA rearrangements. Depending...approximately 20–50% of the packaging cells after 1–3 days of selection. A lower percentage of surviving...up-to-date tips for your virus experiments. References 1. Anthony, Simon J., et al. "A strategy to estimate...experiment and they don't do anything. Viruses are complicated and they perform complex biological tasks, both...vector may undergo genomic rearrangements during replication. To avoid genomic rearrangements, try amplifying...membranes (Davis et al., 2002). Polybrene is one such cationic reagent that is commonly used and has been shown... during transfection, and does not necessarily indicate the production of virus. Alternatively, if the...

...cell with the images from the Allen Cell Explorer (1). When studying the function of a protein or its role...various subcellular structures. Please see Figure 1 for commonly used markers for major mammalian organelles...Subcellular Localization Poster here! References 1. Allen Institute Plasmid Pages 2. Cooper GM. 2000....functionality can be gained by understanding the location and transport of the protein within a living cell...to observe membrane trafficking events in vivo. Locate your protein and see who it is hanging out with...potential overlap, it is possible to determine the location of proteins within complex structures. In order...way it is important to choose the appropriate quantification methods and tools. It is usually not enough...

...active for greater than 50 generations. Figure 1: Mobile-CRISPRi's modular components. Image from Peters...on a non-selective plate. Most bacteria tested had 1 transfer in every ~100 to 1,000 colonies, but some...including E. faecalis and P. mirabilis, had only ~1 transfer per 1,000,000 colonies. Higher transfer rates...The vast majority of bacteria are undomesticated which limits the tools scientists can use to study them...the genome and is propagated during normal DNA replication and cell division. Mobile-CRISPRi does not disrupt...single-gene knockdown experiments. Mobile-CRISPRi applications Mobile-CRISPRi expands CRISPRi to non-model ...Elizabeth A., and Pamela A. Silver. "Harnessing undomesticated life." Nature microbiology 4.2 (2019): 212....

.... pH and DNases affect DNA stability Figure 1: So many choices! What to do? The main factors ...your downstream experiment. TE (10 mM Tris-HCl,1 mM EDTA, pH 8.0) buffer is the best buffer for preserving...If you use a kit for DNA purification or if you use a DIY purification protocol, you might have noticed... mutations in single stranded DNA undergoing replication.   DNase on the other hand can digest and degrade...degradation during plasmid DNA extraction and purification from E. coli is the presence of the protein ...most common E. coli strains used for plasmid amplification are endA negative, meaning the gene has been...chelating agents that can be introduced in the purification protocol to remove endonucleases activity. For...

... spread out onto the plate. We test the (capped) 1:1 agarose/DMEM solution against a part of the body ...a 60 mm dish ready for picking is shown in figure 1.   Find Additional Useful Protocols on Addgene's Protocol...to rapidly punch out cell clones with sterile truncated pipet tips. Indeed, an experienced operator can...wiped down with 70% ethanol, and a box of sterile truncated pipet tips (USA Scientific #1011-8410) is also..., and making sure we always maintain a backup replicates of each clone in case of contamination. We do...