...of these neurons via the channel Channelrhodopsin 2 (ChR2) reduced glutamate transmission from the mPFC...deliver Cre-dependent expression of  Channelrhodopsin 2 (ChR2), a modified receptor protein that activates...movement in an open field box or a circular corridor apparatus using a camera or tracking software. Locomotion...the open (risky). Taking advantage of these characteristic traits, rodents can be used to study anxiety...measuring the time spent in a particular part of the apparatus, it turned out that the IL-1 beta overexpressing...

...plasmid kit consists of 20 plasmids encoding 14 alpha, 2 beta, and 4 gamma subunits of the heterotrimeric G...activation via bioluminescence resonance energy transfer 2 (BRET2). Check out our blog post on GPCR signaling...created an open source resource called PRESTO-TANGO (parallel receptorome expression and screening via transcriptional...

...11 and Cas13 are 1). non-specific RNA cleavage and 2). cell toxicity. Cas7-11 does not display collateral...Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell, 163(3): 759-771 (2015). 10.1016...science.aav4294 Ozcan, Ahsen, Krajeski, Rohan, Ioannidi, Eleonara, et al., Programmable RNA targeting with the single-protein...

... that all look exactly alike.      Figure 2: Reagent gradient. Here, the lanes are lightly marked...guide. The gel is then removed from the membrane (2) and the membrane is cut so each lane is its own piece...in the bands, you may want to consult Tables 1 and 2 in our Technical Design of a Western Blot post to ...the antibody’s dynamic range. Specifically, the 1:2, 1:1, and No dilution samples all have approximately...conditions are 4 °C overnight or room temperature for 1–2 hours, there is some evidence that antibodies may ...buffer. You’ll want to run long enough to get good separation in your ladder, particularly in the sizes around...

...scientists have 1) the courage to make mistakes and 2) the integrity to recognize these mistakes quickly...She studies beta-cell function and failure in Type 2 Diabetes. Birmingham, AL, USA 33.5207°N 86.8025°W ...available to people who want to join the dialog and 2) remember that science should not be partisan: everyone...not only believe in the separation of church and state, but also the separation of fact and fiction. Our...

... fluorescent proteins expressed in the specimen.  2. Fluorescence Activated Cell Sorter (FACs) A machine...photostability of your fluorescent protein when using 2-photon microscopy as this hasn't been well reported...PMID: 18703742. PubMed Central PMCID: PMC2606057. 2. Chtanova, Tatyana, et al. "Dynamics of neutrophil... tagged with a fluorescent protein or dye. It separates the cells mechanically in a vibrating nozzle, ...Schaeffer, Marie, et al. "Dynamic imaging of T cell-parasite interactions in the brains of mice chronically...

...extraction solution, runs the protocol, then uses around 2-4ul in each 50ul PCR reaction. The gDNA extracted ...system, developed by the Zhang lab, which has comparable efficiency to the optimized chimeric design but...better for PCR and surveyor. I used DNA polymerase Takara Ex Taq ™ DNA Polymerase for my genomic PCR, but...successfully used Herc II or Kapa Hifi in PCR. Maybe this Takara enzyme is not very robust in this case for EMX1...processing of the crRNA) is also expressed from a separate promoter. In the pre-processing form, the crRNA...

...Joachim ended up at the University of Amsterdam Chapter 2 - Joachim’s Academic and career goals. Career advice...microscopy (University of Amsterdam). He develops, characterizes and uses genetically encoded fluorescent probes...

...green squares, purple circles and blue triangles. 2) An antibody immobilized on a bead binds specifically...protein G, a novel IgG-binding reagent. J Immunol 133(2):969-974. Hjelm H, Hjelm K, Sjöquist J (1972) Protein...controls for an IP. When possible, perform the IP in parallel with both a sample known to express the protein...identify any non-specific interactions, perform parallel IPs with beads only and an isotype control antibody...

...diffusion test or Kirby-Bauer susceptibility test (Figure 2), and requires at least 18-24 hours to obtain results...." London: Wellcome Trust & HM Government (2016). 2. CDC Information Pages on Antibiotics 3. WHO Antimicrobial...antibiotic’ can also be used to describe anti-parasitic drugs and anti-fungal drugs, because ‘antibiotic...Resistance can be intrinsic, meaning a basic characteristic that naturally occurs in the bacterium, and...

...from animals or hybridomas.   Figure 2: (Left) Figure 2: (Left) The light chain and heavy chain genes..., Aldred SF, Bestagno M, Burrone O, Maynard J, Ferrara F, Trimmer JS, Görnemann J, Glanville J, Wolf P...

...due to the presence of additional helper sequences (2-3). As alluded to above, termination and polyadenylation...and translation processes as well.   Figure 2: The conserved eukaryotic polyadenylation signal directs...transcriptional read-through. Chris Voigt’s lab has characterized a set of prokaryotic terminators and deposited...

...from another within a fully stained sample. Figure 2 is an example of the emission spectra for two fluorophores...another during spectral unmixing.     Figure 2: The full emission spectra for BV421 and BV711 on ...experiment, it is important to consider how many parameters you want to use in a given experiment. For very...

...the chromosome, ending replication.   Figure 2: Prokaryotic chromosomal replication. Adapted from...Microbiology and Molecular Biology Reviews: MMBR, 62(2), 434–464. https://doi.org/10.1128/MMBR.62.2.434-464.1998...cooperatively to separate nearby AT-rich regions of DNA. In a test tube, the DNA helix can be separated by DnaA...ATP-bound DnaA, the proteins cooperatively can separate the DNA strands. Created with BioRender.com. ...

...components, see the AAV Plasmid Elements table . Figure 2: Plasmids required for rAAV vector production. Created... pseudotyped with viral capsids from serotypes 1, 2, and 5 display differential efficiency and cell tropism...the central nervous system . Molecular Therapy, 10 (2), 302–317. https://doi.org/10.1016/j.ymthe.2004.05.024...the production of AAV vectors . Microorganisms, 12 (2), 384. https://doi.org/10.3390/microorganisms12020384...retrograde access to projection neurons . Neuron, 92 (2), 372–382. https://doi.org/10.1016/j.neuron.2016.09.021...were discovered as a contaminant of adenovirus preparations, which is how they got their name, but they ...construction, as it was the first to be well-characterized and extensively studied, and has a broad tissue...

...gene activation, with some genes experiencing around 2-fold activation levels. Although other methods have.... This method has been shown to activate genes at 2- to 3-fold higher than SAM, SunTag, and VPR (Zhou ...once), however, SAM exhibits activation levels comparable to other popular activation methods (VPR and ...

... digest and compare it to some reliable controls. 2. Viral stocks may be sensitive to freeze thaw cycles...PMID: 24003179. PubMed Central PMCID: PMC3760253. 2. Rohwer, Forest. "Global phage diversity." Cell 113.2...10634178. 8. Murnane, John P., Michael J. Yezzi, and Barbara R. Young. "Recombination events during integration...

...mean you have one copy of the gene; an RQ value of 2 means you have two copies, and so on and so forth....quantitative PCR. Expert Rev Mol Diagn. 2005 Mar;5(2):209-19. doi: 10.1586/14737159.5.2.209. PMID: 15833050...falciparum multidrug resistance-1 gene (pfmdr1). J Parasitol. 2011 Oct;97(5):939-42. doi: 10.1645/GE-2792.1...

... unknown Monomer with a valency of 2 IgE Protects against parasites and is responsible for driving allergic...such as anaphylactic shock Monomer with a valency of 2 IgG The most abundant isotype in blood, also found...secondary immune response Monomer with a valency of 2 IgG1; IgG2; IgG3; IgG4 IgM Produced during the primary... ranging from antibody fragments (shown in Figure 2) to completely unrelated molecules. Examples include...used for assembling protein nanostructures. Figure 2: An antibody (IgG, far left) compared to alternative... can be used in the lab to quantify, identify, separate, and locate proteins of interest. The protein ...use antibodies to identify denatured proteins separated by size on an SDS-PAGE gel. Though relative quantification...

...pHelper: 1 RepCap OR 3 Transfer Plasmid : 5 pHelper: 2 RepCap Select what type of tissue culture dish you...goes, “failing to plan is planning to fail.” Preparation is a key step to any experiment and can help ...

...Addgene swag. Good luck and happy naming!   Fig. 2: The mascot trio!     ...our new antibody mascot, who's joining us in preparation for our upcoming affinity reagent distribution...

...Halloween is only 2 days away and here at Addgene we couldn’t be more excited! As a small, tight-knit...(old board games and movies can provide great character costumes). All of this costume planning is for...

...either (1) a lack of gRNA and/or Cas9 expression or (2) a lack of efficient target cleavage in cells expressing...present in a single lentiviral transfer vector or separate transfer vectors May contain reporter gene (e....

...expressed as a fusion with your favorite protein and (2) a synthetic fluorophore-contianing “ligand” that ...cell 58.4 (2015): 644-659. PubMed PMID: 26000849. 2. Grimm, Jonathan B., et al. "A general method to improve...: 26575238. 5. Crivat, Georgeta, and Justin W. Taraska. "Imaging proteins inside cells with fluorescent...

...connectivity to other data sources used in research; (2) connectivity to other tools used in research; and...by making it easy to get data out of the ELN; and (2) facilitate inter-operability with other research ... delve deeper and look specifically at the characteristics and capabilities an ELN needs to facilitate...

...blotting  In a western, proteins are: (1) separated by size, (2) transferred to a membrane, and  (3) detected...system-specific sandwiches.   Figure 2: The western blot dry apparatus includes a stack of filter paper, ...range to be separated on a single gel.  Immobilizing proteins on a membrane Once separated, the proteins... process. The sample, a mixture of protein is separated in a gel, transferred to a membrane, stained with...tertiary structures remain intact and the protein is separated through a matrix by charge. In a denaturing western...protein is denatured to its primary structure and separated by size with smaller molecules moving more quickly... we will be focusing on denaturing westerns.  Separating a protein mix by size Sodium dodecyl sulfate-...

...would label everything but the CD8+ T cells (Figure 2). Even better, we can use the same fluorophore for...gating on the negative population.     Figure 2: Dump gating labels all unwanted cell lineages with...conjugated to a biotin tag but not a fluorophore. In a separate step, cells are incubated with a fluorophore-conjugated...the intranuclear ones. You do not need to use a separate fix/perm cytosolic procedure in that case.   ...cell population. To detect non-surface markers, a separate intracellular labeling protocol is necessary, ...

...to a specific area at the plasma membrane (Figure 2). Just express the plasma membrane-localized iLID ...cells will be ready for the spotlight.    Figure 2: Opto-RhoGEFs to control Rho GTPase activity. A) Schematic...cytoskeleton. They provide cells with physical structure, separate chromosomes during mitosis, support transport ... called a katana. Opto-katanin consists of two separate blue light-sensitive systems, VVDfast to control...your protein of interest and express the other separately, then use blue light to generate CRY2-CIB1 oligomers...

...melanogaster." Molecular and cellular endocrinology 215.1-2 (2004): 1-10. PubMed PMID: 15026169. Soo, Rochelle...algae host for reproduction. Snail parasite Leucochloridium is a parasitic worm that infects a snail’s eyestalks...Brusca and Gilligan, 1983). Grasshopper parasite The parasitic worm Spinochordodes tellinii‘s larvae makes...ve also carved pumpkins (yes, Blugene made an appearance there too) and shared fascinatingly creepy science...Tongue-eating louse Cymothoa exigua is a marine parasite that enters fish through its gills. It attaches...replacement in a marine fish (Lutjanus guttatus) by a parasitic isopod (Crustacea: Isopoda)." Copeia 1983.3 (1983...

...backbone with MS2 loops at tetraloop and stemloop 2 and EF1a-zeo resistance marker pLenti CMV/TO GFP-Zeo...Tet) Inducible Expression page Bacteria Lac, T7, araBAD, trp mTagBFP2-pBAD - Protein expression vector ...astrocyte-selective expression in AAV Dual promoter Separate expression pRGEB31 - Deliver sgRNA and Cas9 into...

... a template, copying the base edit. Figure 2: Cytidine deamination takes place on the free strand...SpCas9 base editors with editing windows as small as 1-2 nucleotides. To reduce off-target effects associated...formation. To further decrease indel formation 1.5-2 fold, the team fused bacteriophage protein Gam to ...K., Arazoe, T., Yachie, N., Banno, S., Kakimoto, M., Tabata, M., Mochizuki, M., Miyabe, A., Araki, M.,...., Slaymaker, I. M., Yen, J., Young, L. E., & Ciaramella, G. (2020). Directed evolution of adenine base...Schwaegerle, N. D., Peraro, L., Young, L., Lee, S.-J., Ciaramella, G., & Gaudelli, N. M. (2023). Improved cytosine...., Hara, K. Y., Shimatani, Z., & Kondo, A. (2016). Targeted nucleotide editing using hybrid prokaryotic...

...environmental contaminants.  Figure 2: An Addgenie works in the tissue culture hood while wearing...doi.org/10.1290/1071-2690(2002)038<0086:caeomi>2.0.co;2 Uphoff CC, Drexler HG (2011) Detecting Mycoplasma ...of the causes of contamination. If possible, a separate lab should be made available for cell culturing...keeping them in the pockets of your lab coats. Have separate footwear for the lab to avoid bringing in environmental...wide range of mycoplasma. These methods cannot guarantee complete removal of mycoplasma but, can get rid...made permanent damage to the cell culture it is irreparable. Some cells may die due to longer incubation ...Microbiol .44: 42–49  Uphoff Cc, Drexler Hg (2002) Comparative Antibiotic Eradication of Mycoplasma Infections...

...wheat germ, HeLa, and Leishmania (Gagoski, et al 2015[2]). The proteins encoded in this set range in size ...to generate Illumina sequencing libraries using a 2-step PCR-based protocol and are compatible with single-end...centered within GFP/RFP bicistronic reporters characterized in Cambray et al., 2013. These terminators can...size from 4 to 156 kDa, enabling the user to characterise the efficiency of their cell-free system in correlation...

...tagging." Nature methods (2015). PubMed PMID:26322837. 2. Stadler, Charlotte, et al. "Immunofluorescence and...the fusions had some negative effect on these parameters. IT5 and IT6 displayed good binding efficiency...

...Agrobacterium infection. At approximately 7 kb, pSiM24 is 2 and 6 kb smaller than commercially available pCAMBIA...duplicated enhancer domains and characterized in studies of tobacco, Arabidopsis, and corn. For both β-glucuronidase...plant infection, as well as plasmid yield during preparation in E. coli. Addgene depositor Indu Maiti has ...functions in multiple species, including tobacco and Arabidopsis. Improving Agrobacterium binary vectors Vectors...efficiently inherited in both transgenic tobacco and Arabidopsis. Finally, Sahoo et al. demonstrate that this ...

...elution choices: TE, Tris buffer, or water Figure 2: Tris-EDTA, Tris, or water? The buffer you choose ..., destabilizing the molecule and favoring the separation of the two helixes. Low pH instead results in... buffer for preserving the stability of your preparation for a long time. Tris buffer controls the pH,... if the priority is to directly use your DNA preparation in any downstream application involving enzymatic...buffer is that it allows better control of the pH preparation stabilizing the plasmid, and still allowing most...at -20°C or even at -80°C, in this case your preparation can be eluted in water or in your buffer of preference...

...blood are reprogrammed immediately after collection [2]. PBMCs display unique epigenetic signatures that ...PMID: 27882265. PubMed Central PMCID: PMC5118044. 2. Kim, Y., et al., The Generation of Human Induced ...vectors encoding reprogramming factors behave as separate extrachromosomal elements in the nucleus of the...

...sequence is unique compared to the rest of the genome.  2) The target is present immediately upstream of a Protospacer...utilized allowing for PAM flexibility. Fig. 2: Prime editor engaging target DNA. The reverse transcriptase...Walton et al., 2020). Further, this group also characterized a second Cas9 variant, SpRY, which cuts at NRN...

...faster when using a dual guide system.   Fig. 2: Size screening for large deletion events.   ... Biol 17: https://doi.org/10.1186/s13059-016-1012-2 Find more CRISPR Resources at Addgene: Read our CRISPRGuide...visualized by running your PCR on a gel. It’s still paramount that you sequence clones to verify that the deletions...

...Almo, Institute for Protein Innovation  1:18:45 - 2:01:18 (end) - Panel Alex Tucker (Ginkgo Bioworks)...their curation of citable, linkable figures. Cause #2: Hidden “negative” data We all want to be able to ... and here I summarize what I learned both in preparation for my talk and at the minisymposium. You can...address this issue directly by creating well characterised antibodies against all human extracellular proteins...stories and read papers wherein a researcher tries 5 separate experiments at once to get confirmatory data for...