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...project has received funding from the Innovative Medicines Initiative 2 Joint Undertaking under grant agreement...

...constitutive overexpression of FPs and tagging of genes of interest with FPs in yeast. Characterization ...

...editing systems include targeted AID-mediated mutagenesis (TAM) from the Xing Chang lab and CRISPR-X from...

...targeting specific neuron subtypes in Cre-driver lines. These could also be used in conjunction with Cre-enhancer...

.... Since this starter cell now has all the viral genes present, the virus can spread from this cell to ...

...pairs bridge the presynaptic and postsynaptic membranes at neuronal synapses. Image credit: IPI Browse...

...may ship at room temperature. However, Addgene determines ideal shipping conditions for each order based...

...2019 Aug 28;5(8):eaaw1822. Scheeren and Verdoes Backbones for mouse recombinant antibodies A panel of recombinant...

...catabolic processes including autophagy and lysosome biogenesis. mTORC2 does not respond to nutrients, but it...

... account. We will notify you by email within 1 business day regarding your account verification. Once ...

...in between a protein and a tag, such as an NLS or NES, enables control over localization. Inserting PhoCl...acrIIA1 Listeria monocytogenes  pRAU168  No  pJH372  No  acrIIA2 Listeria monocytogenes  pRAU171 Partially...  Listeria monocytogenes  pCSW65 N/A (Toxic)   None N/A   acrIIA4 Listeria monocytogenes  pCSW21  Yes...quickly and easily generate zebrafish transgenic lines to study neurobiology and neuorodgenerative diseases...compatible cloning sites for easy cloning of your genes of interest. In order to improve the study of specific...induction of structural plasticity in single dendritic spines. The set of CyRFP constructs available at Addgene... used a combination of deliberate and random mutagenesis to isolate the best RFP. They targeted known ...

...Long Overexpression Tools These AAV encode various genes for overexpression. ID Name Expression System Activity...

... plasmid encoding Rep and serotype-specific Cap genes, and a plasmid encoding adenoviral helper sequences...

...combination with Cre-dependent transgenic mouse lines or co-infection with Cre dependent viral vectors...

...WiChr mScarlet Constitutive 9 Peter Hegemann , Johannes Vierock 154948 CaMKII-somBiPOLES-mCerulean CamKII...

...H., Wong, T. Y., Yang, G. S., Ypsilanti, A. R., Jones, S. J., Holt, R. A., Goldowitz, D., Wasserman, W...

...However, polybrene is toxic to some cell lines. In these cell lines, substitute protamine sulfate for polybrene...the RNAi consortium for targeting human and mouse genes. Protocols... directed against 15,000 human and 15,000 mouse genes. Addgene is working with the TRC to make this shRNA...that can be accessed after a free registration. Guidelines for designing siRNAs with effective gene silencing...polymerase III. Note that these were the prevailing guidelines when the pLKO.1 vector was created. Additional... least 3 nucleotide mismatches to all unrelated genes. TIP: Addgene recommends that you select multiple...Vector The following steps were the prevailing guidelines when the pLKO.1 vector was created. For alternative...

...container Fire blanket Fire extinguisher Guidelines BSL-1 Guidelines Before You Work Right after entering ...chemical hygiene officer. BSL-2 Guidelines Remember, the BSL-1 laboratory guidelines above are expected to be...These lab safety guidelines provide steps to ensure you are working in BSL-1 and BSL-2 labs safely. Protocols...be followed in addition to BSL-2 guidelines below, including PPE protocols . Working in a BSL-2 laboratory...

... but can be adapted to a variety of target cell lines and selection markers. Note this assay requires ...Millipore TR-1003-G 1X PBS pH 7.4 without calcium or magnesium, Corning 21-040-CV (cations can affect the attachment...transduction efficiency. Titer will vary between cell lines. It is not recommended that lentiviral supernatants... to multiple freeze-thaw cycles. This protocol outlines the seeding of the cells at the same time as the... the virus-mediated transduction. For some cell lines, transduction is optimized if the cells are seeded...

...to support reproducibility in science. General Guidelines Acrylamide percentage varies in SDS-PAGE gels...Nonfat milk powder 96-well microtiter plate Chemiluminescence substrate Plastic wrap Primary antibody Secondary... at RT on a shaking platform. Prepare the chemiluminescence substrate by mixing 1:1 reagent A to reagent...reagent B. Gently incubate the membrane in the chemiluminescence reagent for 5 min at RT . Cover the membrane...with clear plastic and use a gel imager with chemiluminescence detection or a dark room to detect the bands...

...hundreds of µL, the second is tens, and the third is ones. Therefore 100 µL would read as 100 from top to ...P20: The first digit is tens of µL, the second is ones, and the third (red) digit is tenths. Therefore,...2.2 µL would read as 022. P2: The first digit is ones of µL, the second red digit is tenths, and the third...at the bottom. For the P1000, this represents the ones. For the P200, this represents the first decimal...

...Then streak horizontal lines in another section of the plate, and then diagonal lines in another section ...very popular technique is to draw in discontinuous lines. Start by streaking a vertical line of bacteria ...

...DNA in other lanes contaminating your sample. To accomplish this, it is best to skip lanes between samples...couple simple ways to increase the resolution (crispness) of your DNA bands include: a) running the gel...

...restriction enzyme -based cloning to test individual clones before use of more expensive forms of plasmid verification... want to know how to differentiate between two clones that differ only in the orientation of the insert...Simply run the digest products on a gel and those clones with the appropriately sized products should have...

...Addgene's Business Development Manager, discusses his transition from the Scientist Team to Business Team,...CRISPR Generate genomic deletions in mammalian cells lines using CRISPR/Cas9 Genomic Deletions with CRISPR ...

...the RNAi consortium for targeting human and mouse genes. Virus Protocols for titering and testing your virus...antibiotic resistant colonies Generating Stable Cell Lines with Lentivirus Genomically integrate your DNA sequence...Population by Limiting Dilution Generate monoclonal cell lines from a polyclonal pool of stable cells AAV Production...

... but can be adapted to a variety of target cell lines and selection markers. Note, because this protocol...Millipore TR-1003-G 1X PBS pH 7.4 without calcium or magnesium, Corning 21-040-CV (cations can affect the attachment...transduction efficiency. Titer will vary between cell lines. It is not recommended to subject lentiviral supernatants...

... can be modified for alternative packaging cell lines or transfection reagents. Once produced, lentivirus...Heat-inactivated FBS 1x PBS pH 7.4 without calcium or magnesium, Corning 21-040-CV (cations can affect the attachment... cells in a T75 flask in 15 mL DMEM Complete. Wednesday: Plate 1×10 6 cells in a T75 flask in 15 mL DMEM...

...migrate through the gel more quickly than longer ones. Thus, you can determine the approximate length ...you can infer the size of the DNA in your sample lanes. For more details on doing diagnostic digests and...A few simple ways to increase the resolution (crispness) of your DNA bands include: a) running the gel...

... to the appropriate well. Pro-Tip Leaving clear lanes between samples will make quantifying the gel easier...Gels . Select Select Next Lane . Repeat for all lanes. Plot the area under the curves for the protein ...Figure 3 Select Analyze . Select Gels . Select Plot Lanes . One graph per lane will appear with peaks representing...

...Reagents Adherent HEK293T cells (ideally AAV-293T clones) Pro-Tip While adherent, these cells are very loosely...Fisher, 12605010) 1X PBS pH 7.4 without calcium or magnesium, Corning 21-040-CV (cations can affect the attachment... + 60 mL of 5 M Sodium Chloride + 4 mL of 1 M Magnesium Chloride Close the bottle and mix by inverting...

...short region of DNA: guide RNAs targeting different genes in an organism's genome, for example. Amplification...altering the composition of the library. Bottlenecks, fitness differences, and plasmid recombination can all ...being lost from the pool and/or increasing the skewness of the pool. 8 electroporation cuvettes (BioRad...

...These guidelines summarize the proper personal protective equipment (PPE) for BSL-1 and BSL-2 labs....Gloves Eye protection and face shields, as needed Guidelines Your lab coat should be buttoned to protect your...

...This approach can be adapted for different cell lines and different transfection reagents. Workflow Timeline...cells in a 75 cm 2 flask in a volume of 15 mL. Wednesday: Plate 1x10 6 cells in a T75 flask in a volume...

... fluorescent secondary antibody. This protocol outlines the steps for fixing and labeling HeLa cells for...and follow your institution's laboratory safety guidelines for disposing of waste in the appropriate container...

...dissociation of the nucleoprotein complexes. The effectiveness of your RNA isolation will depend on how effective...

...are (i) brightness, (ii) photostability and (iii) aggregation tendency. Brightness Brightness refers ...molecule. A higher brightness increases the detected signal and therefore high brightness is an important...how the brightness is best defined or determined. Here, we will explain how practical brightness is determined...determined and why practical brightness is a better criterion than theoretical brightness for selecting the brightest...protein. Theoretical brightness Two key properties of fluorophores that determine brightness are the extent ...number, the higher the theoretical brightness. The theoretical brightness often appears in tables with fluorescent...and the sample. Practical brightness A protein with a high intrinsic brightness that does not fold well ...

... the Lindberg Lab we often make cell lines that overexpress genes of interest; more recently we have also...CRISPR vectors to generate cell lines with knockouts of specific genes. Many years ago, people in the ...give rise to single clones, and thus many plates must be examined for single clones and then handled. Additionally... Additionally, many cell lines, especially the endocrine cell lines we most commonly work with, require...cell clones with sterile truncated pipet tips. Indeed, an experienced operator can pick 48 clones from...individual cell clones with different expression characteristics from a population of transfected clones. The technique...number of single clones obtained (on occasion we have prepared two 48-well plates of clones) which enables...

...targeting their genes of interest to generate mAID fusions to endogenous genes. Cell lines with the appropriate...targeting a total of 20,121 genes within the human genome (the identity of the genes and target sequences can...continuous expansion of compatible backbones, promoters, and genes available to the community. If you ...identify both core and context-dependent human fitness genes, the laboratory of Jason Moffat at the University...cell models and identified ~4-5-fold more fitness-related genes than have been found in previous screens...uncharacterized genes that are conserved among apicomplexans and important for T. gondii fitness. Sixteen of...enable researchers to screen the entire genome for genes that regulate a wide variety of phenotypes, including...

..., which genes are good candidates for being involved in the studied phenotype. For robustness of a screen...presenter then showed which genes they had generated knockout mice for (all of the genes involved as I recall...differentially expressed genes that are known drug targets, or all differentially expressed genes that code for ... if genes that you asked for were not found in the reference library. It’s not uncommon that genes have...5 guide RNAs (gRNAs) targeting each of the 1000 genes you’d like to investigate in your next CRISPR screen...CRISPR screens targeting a (long or short) list of genes provided by the user. This approach is useful for...investigator generates a cell population where all genes are knocked out at the population level, but each...