...overlap with the negative control population (Figure 1). Based on where the GFP- population ends, you can...total cells present in the gate.   Figure 1: Example of yes-no flow plot of the (negative) control...for a yes-no dot plot, like the one shown in Figure 1.  Figure 3: An example of a histogram plot ... Figure 2: (A) a smeary dot plot showing a range of antibody readouts. (B) A gate on the smeary dot...histogram plot for a smeary antibody readout. The orange peis the baseline expression of the protein of ...yes-no histogram plot shown previously. Here, the orange peak is a baseline expression of a protein while...

...reserve a small aliquot, split into two parts to (1) run a DNA gel to confirm fragment size and (2)  incubate...with a phenol-chloroform extraction.    Fig. 1: Schematic overview of ChIP. Image courtesy of Shengliu... let’s do a quick review. At this point you have (1) crosslinked your proteins of interest to any DNA ...Fragmenting  The next step is break the DNA into random, small pieces of roughly equal size, typically ...enzymatic digestion. Sonication provides a truly random digestion, but is harsher and requires more optimization...your protein of interest is a low abundance transcription factor or cofactor. In such cases, the gentler...

...dual-color control of neurons. Nature Communications, 12(1), 1–20. https://doi.org/10.1038/s41467-021-24759-5 Zhang...to consider when choosing one.     Figure 1: Example of an experimental optogenetic setup. A light... 21–51). Springer US. https://doi.org/10.1007/978-1-0716-0755-8_2 Lehtinen, K., Nokia, M. S., & Takala... 21–51). Springer US. https://doi.org/10.1007/978-1-0716-0755-8_2 Boyden, E. S., Zhang, F., Bamberg, E... charged ions to rapidly pass through the cell membrane and initiate an action potential - a very useful...and close when exposed to light and change the membrane potential. OptoXRs are modified GPCRs. When exposed...downstream signaling pathways that can change both the membrane potential and a host of other cellular functions...

...control exactly in which locus your transgene ends up (1): Random insertion has the advantage that you...pound symbol”. References 1. J. R. Bateman, et al. Site-Specific Transformation of Drosophila via PhiC31... you’d like to use to generate your new fly line: 1. Where would you like the gene to be incorporated ...you need to combine multiple transgenes in one fly, having the transgenes in different loci allows for...use? Random insertions take longer because you need to determine in which chromosome your transgene has...brief introduction as to how you can engineer new transgenic flies to study your favorite gene (YFG).  Sometimes... is thrillingly new, you might have to make a transgenic fly in order to study it. The process is fairly...

... emailed submission) in the Deposit menu: Figure 1: Begin the deposit process by clicking "Submit Online...cases Please contact us at [email protected] or +1 (617)-ADDGENE (233-4363) if your deposit falls into...Addgene?" or contact us at [email protected] or +1 (617)-ADDGENE (233-4363) with additional questions...strain. Please contact us at [email protected] or +1 (617)-ADDGENE (233-4363) if you’d like to distribute...page so that any data you enter is not lost.   Step 1: Sequences, Maps, and Files Please provide any full...a 1.5 mL microfuge tube at a concentration of 0.1–1 µg/µL (or bacterial streaks, if you prefer). If you...number of unique plasmids in a pooled library can range from a few hundred to millions. Pooled libraries...

...indicates the presence of a multimer.   Fig. 1: The monomer of Plasmid 12259: pMD2.G (depicted on...plasmid sequencing services using long-read sequencing (1-25 kb), such as Oxford Nanopore sequencing or PacBio...10017-26. doi: 10.1074/jbc.M110.176636. Epub 2011 Feb 1. PMID: 21285356; PMCID: PMC3060453. Bazaral M, Helinski...multiple circular DNA forms of colicinogenic factor E-1 from Proteus mirabilis. Biochemistry. 1968 Oct;7(10...Methods Mol Biol. 2009;582:11-25. doi: 10.1007/978-1-60761-340-4_2. PMID: 19763938. Maucksch C, Bohla A...size, multimers may exhibit reduced transformation and transfection efficiency compared to the monomeric... Elfinger M, Hartl D, Rudolph C. Transgene expression of transfected supercoiled plasmid DNA concatemers...

... tissue. References 1. Aschauer DF, Kreuz S, Rumpel S. Analysis of transduction efficiency, tropism and... and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain. PLoS One. 2013 Sep 27;8...M. Human synapsin 1 gene promoter confers highly neuron-specific long-term transgene expression from an...AAV5, AAV8) enable different patterns of transduction and transgene expression across different cell types...expressed from your cell line of choice after transduction with this virus. While various Cas9 mutants ...classic Cas9 nuclease, which introduces double stranded DNA breaks, can be expressed from lentiCas9-Blast...single stable Cas9-expressing cell line and then transduce this line with lentiviruses expressing various...

...-HA frankenbodies include mEGFP, mCherry, and HaloTag. Figure 1: (a) Anti-FLAG frankenbody (FB)...Comm. 2019. https://doi.org/10.1038/s41467-019-10846-1.   Optimized genetic screening technique using Cas12a... to Cas9’s larger expression requirements (Figure 1), but Cas12a has generally been underutilized in genetic...Here's what you'll find in this post: Anti-FLAG Frankenbodies for modular imaging of cellular targets Optimized...mammalian genome regulation and editing Anti-FLAG Frankenbodies for modular imaging of cellular targets by: ...: Mike Lacy Adding to their set of anti-HA frankenbodies (Zhao et al. 2019), Tim Stasevich and Hiroshi... have recently published a set of anti-FLAG frankenbodies, a flexible, modular tool for labeling. Here...

...unleashes its cargo – its own genome.   Fig. 1: Viral particle infection into a host cell. Image ...Vector Resources at Addgene! References and Resources 1. Roe T, et al. “Integration of murine leukemia virus...nuclear import of human immunodeficiency virus type 1 preintegration complexes.” Proc. Natl. Acad. Sci. ...one they have a high transduction efficiency and can effectively infect a wide range of cell types that ...used to achieve transient, high expression of the relevant cargo without the fear of random genomic integration... Packaging Capacity (>8kb) High Transduction Efficiency Transient Gene Expression High Immunogenecity...viral envelope, which fuses with the target cell’s membrane, allowing the viral particle access into the cell...

...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...observational study. The Lancet Microbe 1:e14–e23 . https://doi.org/10.1016/s2666-5247(20)30004-5 McCray...scientific community. Addgene was awarded a Fast Grant to ensure that the scientific community will continue...-2. The resurgence in popularity of the hACE2 transgenic mouse strain, for example, is sustained by the...University of Iowa (McCray et al., 2006), the hACE2 transgenic mice were designed after it was discovered that...Deposits of new reagents, policies from journals and granting agencies requiring materials to be deposited into...

...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 ... different cell types so it is useful in a wide range of applications. This plasmid was the top requested...pSpCas9(BB)). The 2A-GFP allows for screening of transfected cells. Find pSpCas9(BB)-2A-GFP (PX458). lentiCRISPR...lentiCRISPR v2 - This 3rd generation lentiviral transfer vector also from the Zhang lab is used to deliver...David Root lab is a 3rd generation lentiviral transfer plasmid for cloning and expressing new shRNA sequences...the necessary elements for packaging, reverse transcription, and integration needed for the production of...

... the samples stored in our lentivirus tubes (Fig. 1). However, once thawed, there was a substantial reduction...in the pH of samples stored in our AAV tubes (Fig. 1). It’s important to note that the change in pH only...using the thawed virus within 24 hours. References 1. Murphy BM, Swarts S, Mueller BM, van der Geer P, ... However, the pH changes in our solutions are transient and return to normal once the tube is opened and...restored. We have no evidence to suggest that the transient change in pH affects AAV function. Furthermore...Furthermore, another study tested AAV infectivity after a range of pH exposures. They showed that AAV infectivity...MC, Fitchmun MI. Protein instability following transport or storage on dry ice. Nat Methods. 2013 Apr;10...

...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...edit. Since exploitation of NHEJ to generate translational reading frame-altering indels for “functional... an unedited site distal tothe predicted double strand break, and a NHEJ/ “drop-off” probe designed to...to bind directly at the predicted double strand break site (Figure 2A). The single molecule resolution...PubMed PMID: 26914317. 4. Mock, U. et al. mRNA transfection of a novel TAL effector nuclease (TALEN) facilitates...

...flanked by ~50-150 bp transposon end sequences (R and L ends).  Figure 1: (A) Components involved...CRISPR-associated Transposase) by the Zhang group. Mechanism of CRISPR-transposons One of the CRISPR-transposon systems...DNA transposons have been hindered by their limited programmability: depending on the transposases involved...-guided transposition. By natively combining the DNA integration capabilities of transposases and the ...crRNA.  Three transposase proteins (TnsA, TnsB, TnsC). The donor DNA (aka mini-transposon), which contains... each type of CRISPR-Transposon system. (B) Mechanism of RNA-guided transposition in type I-F INTEGRATE.... Separately, TnsABC forms a transposase complex with the mini-transposon, excises it from the donor molecule...

...skeletal muscle contraction.   References 1. Degorce, François, et al. "HTRF: a technology tailored for..."Förster resonance energy transfer measurements of ryanodine receptor type 1 structure using a novel site-specific...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. ...they cannot cross cell membranes, so they are best used either with plasma membrane proteins (such as GPCRs... minimum energy transfer values you can expect, thereby establishing the dynamic range of your FRET assay...

... 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 ...meeting at both the Fox Chase Cancer Center and Brandeis University. 5 easy steps to forming your own peer... of people who are all interested in making a transition. Schedule your first meeting and get organized... your successes and support each other as you transition to the next stage in your careers! Stay in touch...

...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... technologies have transformed the palette of experiments possible in a wide range of organisms and cell...genomic locus (CRISPR-ChAP-MS) could further transform the range of experiments imaginable in C. elegans. ...

... levels. That is, if transcription factor 1 controls expression of transcription factor 2, which directly...by modulating the induction of transcription factors 1 and 2. See Aranda-Díaz et al. for more information...difficult-to-manipulate primary cells, achieving knockdown within 1-2 hr of reagent delivery. TRIM-Away is suitable for...expression, Aranda-Díaz et al transformed yeast with both a GAL4/estradiol controlled transcription factor ... a broad pH range. The four biometric probes successfully targeted endosomes under a range of pH conditions...ligand binding domains and the MSN2 transactivation domain. Transcription factor activation via the appropriate...progesterone controlled transcription factor as well as reporters specific to each transcription factor. They then...

... help improve the transport of protein of interest the membrane.   Figure 1: Electron microscope...proportion were found on the plasma membrane (top panel in Fig. 1). Of course, this was problematic, because...expression of hM4Di in plasma membranes in both species (bottom panel in Fig. 1). Our results indicate that... the transgene of interest. In our studies, mCherry interfered with the transport to the membrane of the...the hM4Di might improve the transport of this DREADD to the plasma membrane. The haemagglutinin (HA) tag...the expression of the transgene of interest and its localization in the membrane. The lesson we learned... the plasma membrane. Note that the use of the HA tag significantly improves the transport of hM4Di to...

... Figure 1: Strand separation and target DNA binding to the Cas9 nuclease. Stable strand separation ...by 2 sets of interactions : 1. Interactions between the non-target strand and a positively charged groove... improvements in genome engineering! References 1. Slaymaker, Ian M., et al. "Rationally engineered ...increases when target DNA strand separation is stabilized (2,3). Stable strand separation is maintained...and therefore destabilize basal strand separation. This decreased strand separation combined with the weak...the non-target strand and the HNH/RuvC groove, and interactions between the target strand and the gRNA....nuclease domains 2. Interactions between the target strand and the gRNA (see figure below) Because off-target...

...Figure 1: The Addgene Requests Dashboard   The dashboard will display the following items (Fig. 1): ...depositor, but it can also be valuable data to share in grant proposals or to demonstrate impact in annual reports...

...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 ... that determine membrane expression and plasma membrane localization of transmembrane proteins, like rhodopsins...capable of broad transduction of the nervous system which eliminates the need for intracranial injections. ... to perform in vivo optogenetic experiments. Intracranial injections of AAV are commonly used to deliver...channelrhodopsin-based optogenetics minimally-invasive, Frances Arnold’s lab and Viviana Gradinaru’s lab used machine...

...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... copy. The coding sequence for GFP is transcribed and translated along with the gene, producing both a...exon of the gene to ensure it would be transcribed and translated. For an N-terminally tagged protein, ...destroyed by CRISPR/Cas9 during transfection. Making the double strand break and introducing the tag through...base pairs on each side, in fact. Later, when we transfect the cells, these large regions of homology enable...

...Popular gamma-Retroviral Transfer Vectors   Expressing your gene of interest pMKO.1 GFP (Plasmid #10676) ...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....split-vector systems’ by separating viral genes and transgene expression across several plasmids. The most commonly...separate envelope and packaging plasmids as well as transfer plasmids. This concept ensures safe handling and...Gamma-retroviral vectors have a relatively high transduction efficiency, but in contrast to lentiviral vectors... are very versatile tools as they are able to transduce dividing and non-dividing cells. More detailed...

... biosensor, so that the (1) binding of calcium, (2) allosteric rearrangements of the sensor, and (3) subsequent... to a red fluorescent state only by simultaneous (1) user-controlled illumination with violet light and...fluorescent indicators. Nature protocols. 2006 Aug;1(3):1057-65. Resources on Addgene.org Addgene’s AAV...your expected range. For example, calcium in the ER lumen reaches the sub-millimolar range, much higher...are used to monitor calcium concentrations and transients in living cells and organisms. Unlike chemical...sensors available that will localize at the plasma membrane. GCaMP-X is a nucleus-targeted GCaMP6 with an ...an extra apoCaM-binding motif to prevent aberrant binding of the biosensor to endogenous calmodulin. Multiplexing...

...domain (Figure 1).  Figure 1: Examples of sites on antibodies used for conjugation. 1) Endogenous ...microscopy, flow cytometry, or spectrophotometry. The range of fluorophores available for antibody conjugation... choice for ELISAs.  Biotin Biotin is used in a range of antibody-based assays. Biotin itself is not visible...Endogenous carbohydrates added to antibodies post-translationally. 4) Modifications made to antibodies, such ...commercially conjugated antibodies available, that never guarantees the antibody you need will be conjugated, or ...Stephenson W, et al (2017) Simultaneous epitope and transcriptome measurement in single cells. Nat Methods 14:...

...current state (Green when off, dark when on). Figure 1: Photoswitchable MEK1 (psMEK1). Is it possible to ...Below are a few examples demonstrated in Zhou et al. 1. Testing kinase inhibitors Zhou et al examined the...the protein and pdDronpa are preserved. References 1. Zhou, Xin X., Linlin Z. Fan, Pengpeng Li, Kang Shen...Signal transduction pathways are a lot like cell phone networks. Protein kinases deliver messages to ..., and how long their signals last all impact transduction of the message. To study signaling pathways,... game of telephone, often the signal must be transmitted by intermediate messengers. Optical control of...signaling by using psMEK1 and an ERK kinase translocation reporter with a mRuby2 fluorescent reporter ...

...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...Application. Clinical and Developmental Immunology 2012:1–15 . https://doi.org/10.1155/2012/980250  Bates A,...without disulfide bonds, made by molecular evolution 1 1Edited by I. A. Wilson. Journal of Molecular Biology... the CAR facilitates signal transduction and release of cytotoxic granules from the T cell. When a CAR...., 2012, Bates and Power., 2019): rapid blood clearance, which is useful for imaging applications; better...small size (Bates and Power., 2019). Their rapid clearance from blood can be a drawback for therapeutic applications...

...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...expense, and less of being left with a bunch of random solutions when all the spin columns run out. In... inherently not as stable as DNA- it is single stranded and its ribose groups are susceptible to hydrolysis...effectively isolate intact RNA, a fast, strong protein denaturant would be required- something that broke down ...Chirgwin and colleagues showed that a strong protein denaturant, guanidinium thiocyanate, did just this (Chirgwin...

... 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...purification of tagged transcription factors in mammalian cells and transgenic mice.” PNAS 100.13 (2003...and decarboxylases. These enzymes catalyze the transfer of carboxyl groups to organic acids to produce...lipogenesis, amino acid metabolism and energy transduction. Biotinylation is the general process by which...

... 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...strong transcriptional terminator BBa_B1002 (from IGEM) upstream of the MCS to reduce transcriptional readthrough...rely on exogenous enzymes, require multiple transformation and plasmid curing steps, and can generate ...worked too. Rhamnose induction relies on the transcriptional activator rhaS. To allow manipulation of bacteria...

...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...poison, locking up DNA gyrase with broken double stranded DNA and ultimately causing cell death. ccdA, another...gene in frame with an MCS. E. coli that were transformed with the empty vectors expressed the ccdB gene... that ccdB Survival™ can be more difficult to transform, depending upon the specific plasmids being used...

... 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...proceeded through NHEJ rather than HDR. Figure 1: Homology-directed repair in targeted zygotes proceeds...the embryos underwent repair not from the single-stranded oligonucleotide (ssODN) template supplied by Ma...maternal allele rather than the supplied single-stranded oligonucleotide (ssODN) template. Embryos also...off-target effects, but this advantage likely won’t transfer to all genes we’d like to target. In addition,...

... two of the remaining viral proteins. Only Nsp11 ran bigger than expected, and Orf7b gave a lot of background...received so many requests (175 and counting) that we ran out of FedEx supplies at one point and had to go ...faster. Years of science, done in weeks Figure 1: Sending out 80 FedEx envelopes in a single day --...right) from the University of California, San Francisco. Manon Eckhardt and Melanie Brewer are on the ...

...destabilize the transcription complex, initiating cleavage of the transcript.   Figure 1: A predicted... monkey virus constitutive RNA transport element lead to enhanced HIV-1 Gag expression in a context-dependent...prokaryotic terminators and deposited several with Addgene (1). Prokaryotic polyadenylation Although mostly thought...initiation step of gene transcription; today we'll provide an overview on how transcription stops, or termination...from the transcription machinery. Terminators are found downstream of the gene to be transcribed, and typically...generally serves to promote transcript longevity in eukaryotes and promote transcript degradation in prokaryotes...Hepatitis Virus Posttranscriptional Regulatory Element Enhances Expression of Transgenes  Delivered by ...

...Podcast breakdown (Time : Topic) 0:00 - 1:25 : Introduction 1:25 - 6:19 : Dr. Koeris' graduate work in...Novophage's successful transformation into Sample 6 and Dr. Koeris' own transition from graduate student...37-36:53 : Was It difficult for Dr. Koeris to transition from developing therapeutics to developing a ...

... DNA that were transcriptionally active in retinal cell types in mice. Figure 1: The four strategies...used four different design strategies: Strategy #1 (ProA): Create promoters with sequences upstream of...highest rate of targeted expression. Surprisingly, <1% of synthetic promoters replicated the expression ...mouse cell type specific transcription factor binding sites interspaced with random sequences.   Strategy...synthetic promoters. Legend: TSS: transcription start site. TFBS: transcription factor binding site. Image from...particular cell type is important not only for translational research, such as gene therapy, but also basic... using naturally occurring promoters to drive transgene expression, the Roska lab built synthetic promoters...

...yourself in a creative rut, read on!   Fig 1: Blugene using creative problem-solving in the lab...Blog Transferable Skills Guide: Problem-Solving Transferable Skills Guide: Teamwork Transferable Skills...feel they lack formal training in a variety of transferable skills. At Addgene we're helping to fill this...both helping researchers recognize their MANY transferable skills learned while working in the lab and ...Today, we’re highlighting an oft-underlooked transferable skill: creativity. I’ve heard quite a few scientists..., it’s clear that creativity is absolutely a transferable skill developed during PhD training. Creativity...problem. In a study outlined in Originals by Adam Grant, a creativity expert at Stanford conducted an experiment...

...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...fluorescent proteins in the same spectral class (Cranfill et al, 2016). This non-linear dependence on excitation...the conclusions reached at high power may not translate to real applications in which far less power is...

..., including those found at Addgene! Figure 1: Strategy for assembling the barcoded gRNA library...pool-clone them into the pAWp28 storage vector (Figure 1): Forward oligo: 5’- CACCGNNNNNNNNNNNNNNNNNNNGTTTGGGTCTTCGAGAAGACCTATTCXXXXXXXXC... He can be contacted at [email protected].   References 1. Cheng, Allen A., Huiming Ding, and Timothy K. Lu....expression of other genetic elements including transcription factors (Cheng et al., 2014) and microRNAs (.... Konermann, Silvana, et al. "Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex...

...functioning T cell repertoire (1). In this example, it was possible to transduce the T-cells outside the body...significant improvements in vision over a period of 1 to 3 years – the treatment appears to be both safe...research and science communication.     References 1. Gaspar, H. B., S. Howe, and A. J. Thrasher. "Gene...their mutations corrected, and the T-cells were transferred back into the infants. Almost all patients have...techniques may hold the key. In the simplest form, translating gene therapies from conceptual design to clinical...that carries a small, approximately 4.7 kb single stranded genome. AAV belongs to the family Parvoviridae...AAV vectors are also capable of infecting a wide range of cell types including but not limited to muscle...

..., 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... non-specific endonuclease (Cas9) and a single stranded guide RNA (gRNA). The ~20 nucleotide targeting...of wild-type Cas9 and a gRNA generates a double-strand break in the target DNA, which, when repaired through...scaled up for genome-wide screening due to the broad range of potential target sequences and ease of generating...heterogeneous population of gRNA-containing lentiviral transfer vectors, each targeting a specific gene within...

...! References 1. Sinche, Melanie, et al. "An evidence-based evaluation of transferrable skills and job...Page Read Other Posts in the Transferable Skills Guide Developing Transferable Skills During Science Training...that they lack formal training in a variety of transferable skills. At Addgene we've set out to fill this... highlighting that researchers do learn MANY transferable skills while working in the lab and by offering...where you might need some help. Today in our transferable skills guide: Collaborating with others outside... had to overcome some hesitation to ‘approach strangers’ and learn how to communicate outside my scientific...message and to keep it short. Soon, discussing with strangers stopped feeling weird and started to be fun. I...

...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... polymerase and transcription factors. Since the promoter region drives transcription of a target gene...repressor then prevents transcription from occurring, which means that transcription is now OFF.   We’ll...tetracycline-controlled transactivator (tTA). They fused TetR with the transcriptional activation domain VP16...

...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...mutations with unc- 119 + selection." MicroPublication 1 (2018): 2–3. Wang, Han, et al. "cGAL, a temperature-robust...neurons from each class to fully characterize the transcriptome. The first data release for CeNGEN is planned... The 3’UTRs of mRNA are important for post-transcriptional regulation in C. elegans. To study the role...using a split intein for refined spatiotemporal transgene control in Caenorhabditis elegans." Proceedings...

... when (1) other strategies may not be feasible and (2) large-scale proteomic and transcriptomic datasets...expected in another. You’ll need to validate that points 1 and 2 hold true under your specific experimental conditions...application and available resources.     Figure 1: Table summarizing the IWGAV five proposed antibody...validation: A view from the mountains. New Biotechnol 45:1–8. https://doi.org/10.1016/j.nbt.2018.08.002  Uhlen...Interest (GOI), and transfect it into your favorite cell line. In parallel, you transfect cells with another...antigen - Recognition of an immunogen does not guarantee recognition of the target antigen. You want to...Antibody-independent methods may include analysis of transcriptomic or proteomic data from several different tissues...

...and P. mirabilis, had only ~1 transfer per 1,000,000 colonies. Higher transfer rates are better suited for...non-selective plate. Most bacteria tested had 1 transfer in every ~100 to 1,000 colonies, but some bacteria...active for greater than 50 generations. Figure 1: Mobile-CRISPRi's modular components. Image from Peters...protocols can transfer CRISPRi into a single bacterial strain, such as a B. subtilis, or a narrow range of bacterial...can be introduced into a broader range of bacteria. Once transferred, Mobile-CRISPRi stably integrates...bacterial Tn7 transposon system. This system has two parts: a plasmid expressing the Tn7 transposition genes ...chromosomal copy of the RP4 transfer machinery mobilize these plasmids for transfer to recipient cells during...

... (which range between 0 and 100) are then converted to a weight (which ranges between 0 and 1) so that...values (ranging from -40 bp to 40 bp) are also converted to a weight (which ranges between 0 and 1) (shown...distance) This gives each guide a score ranging between 0 and 1. The cutting frequency determination (CFD...summary per batch. Ranking guides for HDR in-depth In other tools, guides are ranked by specificity of ...genome by eye. We came up with a ranking formula that does just this: Rank = (compressed CFD score) * (gaussian...interactions (e.g., scores >80) are transformed to 100. This transformation (illustrated in the graph below...employs biological best practices. For example, it ranks guides by CFD score, and it mutates HDR donor templates...

...activating kinase 1 (MEK1) with either one or two phosphoserine residues cotranslationally inserted in their...directionally assembled into a Level 1 vector creating a single transcriptional unit (Ex: a promoter, 5’UTR, ...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...comprised of three sets of cloning vectors (Level 0, 1, or 2) which can be utilized in three successive assembly...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 ...

...Please see Figure 1 for commonly used markers for major mammalian organelles and transport pathways. It is...cell with the images from the Allen Cell Explorer (1). When studying the function of a protein or its role...Subcellular Localization Poster here! References 1. Allen Institute Plasmid Pages 2. Cooper GM. 2000....can be gained by understanding the location and transport of the protein within a living cell. Analyzing...Analyzing differences in protein localization and transport between healthy and diseased states can also provide...endoplasmid reticulum, modified in the golgi and then transported in vesicles via the secretory pathway to their...the end of their life cycle, proteins may be transported through the vesicles to the lysosome where they...