...genome-wide libraries for CRISPR screening. Sanjana NE, Shalem O, Zhang F. Nature Methods. 2014 Aug;11(8...Protocols Generating Stable Cell Lines with Lentivirus Generating Stable Cell Lines with Lentivirus You may also...Protocol to generate stable cell lines expressing a gene of interest from an integrated lentiviral vector...This protocol can be used to generate stable cell lines expressing a gene of interest from an integrated...transient transfection approaches, generating cell lines using lentiviral vectors enables long-term protein...293T cells but can be adapted to alternative cell lines. Workflow Timeline Day 0: Seed and transduce Cells...needed Day 14–18: Expand and harvest stable cell lines Equipment Class II, Type A2 Biological Safety Cabinet...

...genome-wide libraries for CRISPR screening. Sanjana NE, Shalem O, Zhang F. Nature Methods. 2014 Aug;11(8.... After the monoclonal lines have been sufficiently expanded, screen the lines for transgene expression...transgene expression over time, as the lower expressing clones take over the polyclonal cell pool. Generating ...details, see our protocol for generating stable cell lines with lentivirus . Day 0: (optional) Seed cells for... cells Day 14–30: Analyze and expand monoclonal lines of interest Equipment Class II, Type A2 Biological...Heat-inactivated FBS 1X PBS pH 7.4 without calcium or magnesium, Corning 21-040-CV (cations can affect the attachment...pool: see our protocol for generating stable cell lines with lentivirus . Pro-Tip Because the polyclonal...

...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 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...