...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 Name Description Link to Video General Transfection...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...

...of insertional mutagenesis. Random integration also creates heterogeneous iPSC cell lines, which can complicate...complicate comparisons made between lines. Incomplete silencing of transgenes is a concern as well, and reactivation...reprogramming transgenes and the up-regulation of corresponding endogenous pluripotency genes. Retroviral...to insertional mutagenesis and random integration creates variation between iPSC clones. Retroviruses ...disrupt the expression of tumour suppressor genes and/or oncogenes, potentially leading to the presentation...insertional mutagenesis, the presence of a genetic scar, and incomplete silencing of transgenes. Overall,...Efficient and Stable Genome integration Insertional mutagenesis, transgene reactivation, residual expression ...

...profiles (Dwight, 2011).  Reference Genes  Choosing a set of reference genes deserves more attention than it...want to ensure that your reference genes are suitable for your cell lines and/or tissue types.  Please do...reference genes, plural. Your data will be much improved if you move to using multiple reference genes whenever... multiplex your reference genes to save on plate space. For many cell lines and tissue types, appropriate...and most machines have a protocol that automatically performs the normalization step.  Machines Most general-use...researcher defines log-linear phase Liu and Saint No Raw Data Reference and target genes can have...previously-generated validation data, be aware that different machines, samples, and treatments may affect the assay....

... epitope tagging of a large cohort of genes (e.g. tagging genes representing a protein super family). ...generation of clonal knock-in lines that we now use for IP and ChIP assays. Tag 96 genes at a time using Custom...knock-in for 96 different genes in a single go (Figure 3). We were able to tag >30 genes/plate in the first ...Derive clonal lines by single-cell deposition in 96-well plates: Characterize clonal lines by ICC and PCR...designed for ~250 genes, we have had about 30% tagging success, although many of these genes were expressed...be used at high throughput because numerous cell lines can be generated, each with a different tagged protein...ChIP assays. Creating epitope-tagged clonal cell lines remains a challenge as it relies on inefficient ...

...community – particularly ones that help address ongoing problems, like cells lines. A 2014 study in Science...145,000 cell lines. Cellosaurus Cellosaurus seeks to identify and describe all cell lines used in research...and dog cell lines is included when available, and can be submitted via email for cell lines profiled in...Cellosaurus also identifies problematic cell lines: ones either suspected to be contaminate or easily ...as commonly misidentified lines. Browsing this feature to know if your lines may be especially vulnerable...to help you identify proximal lines you care about. If you have cell lines without a certified origin (...Science found that approximately one-third of cell lines used in scientific research are misidentified! Rigor...