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...stable cell lines Equipment Class II, Type A2 Biological Safety Cabinet 0.5–10 µL single channel pipette...tissue culture treated dish, Corning 3516 15 mL conical tubes, VWR 21008-216 Lentivirus preparation Appropriate...You Start The health of the target cell line is critical for obtaining accurate results. Check the cells...lower dilutions depending on your downstream applications. If you’ve titered your virus beforehand, you...and/or harvest to test for protein expression. Typically, cells transduced with lower dilutions of the ...expressing Cas9. A549 cells were transduced with lentiCas9-Blast and then selected with 1 µg/mL blasticidin...formed by expansion of single cells for 13 days. lentiCas9-Blast was a gift from Feng Zhang (Addgene plasmid...

...Immunocytochemistry Protocol Recombinant Antibody Purification Protocol Antibody Guide Introduction This protocol...primary antibody to use will vary and needs to be empirically determined. If possible, run an initial test ...Bovine serum albumin (BSA), VWR 10273-264 50 mL conical tubes, VWR 89039-656 96-well polyester (clear) ...antibody concentration will vary and needs to be empirically determined. We suggest starting with three concentrations... rpm for 15–30 min . Pro-Tip Check the plate periodically to determine when the desired color change has...display a green product. A precipitated product indicates the presence of too much HRP and the need to optimize...curve will vary between targets and needs to be empirically determined. The dilution series created in section...

...post on ddPCR for AAV quantification. This protocol was modified from the publication Lock et al. (2014) ...Adeno-associated viral vectors (AAV). This protocol specifically uses primers and probes targeting the ITR elements... is genome copies and generally represents the physical number of viral particles. Users may need to run...biosafety regulations. Equipment Class II, Type A2 Biological Safety Cabinet, Labconco 302411100 Droplet Digital...cartridge holder with bleach and place in the Biological Safety Cabinet (BSC). Make sure that the BSC ...Pro-Tip Prepare the viral dilutions in a separate biological safety cabinet (dilution BSC) than that used ...

...larger conical tubes. Depending on the centrifuge their speed ranges can vary, but are typically lower ...Tabletop Centrifuges sit on your benchtop and typically hold the common 1.5 mL microfuge tubes used in...volume as your samples. If your samples are significantly higher density than water, then you may need...according to speed and time (and temperature, if applicable) listed in your protocol. Pro-Tip Spin speed ...centrifuge begins to wobble or shake, this could indicate an unbalanced or improperly loaded centrifuge....

...) to titer lentivirus vectors. This protocol specifically uses primers and probes targeting integrated... sample. This protocol was modified from the publication Wang et al. (2018) . Before Starting Thaw the...ddPCR and analysis Equipment Class II, Type A2 Biological Safety Cabinet Aspirating unit Microcentrifuge...Scientific, 10199-452 Reagents GeneJet Genomic DNA Purification Kit, Thermo Fisher, K0721 6-well tissue culture...the next dilution. Repeat for all dilutions as indicated in the chart below. Dilution Factor Virus Volume...genomic DNA according to the GeneJet Genomic DNA Purification Kit instructions. Determine the concentration...cartridge holder with bleach and place it in the Biological Safety Cabinet (BSC). Make sure that the BSC ...

...pairs) for visualization and purification. Electrophoresis uses an electrical field to move the negatively...eye protection and gloves when working with this chemical. Note: If you add EtBr to your gel, you will also...approximately 75-80% of the way down the gel. A typical run time is about 1-1.5 hours, depending on the...instructions on how to do this, visit the Gel Purification page. Tips and FAQ How do you get better resolution...polyacrylamide gel electrophoresis (PAGE), which is typically used to separate 5 - 500 bp fragments. How do ...

...polymerase that can withstand radical temperature changes during a typical PCR. The DNA polymerase has ...template), a researcher can create thousands of identical copies using a simple set of reagents and a basic...GC-rich templates. What does each ingredient specifically do? Template DNA: Contains the portion of DNA...Buffer with MgCl 2 : Provides an optimal and stable chemical environment for the DNA polymerase to work adequately... provide a suitable environment for the DNA amplification reaction. Reference Page Top Index...

...after working in the lab. Ensure that a designated chemical waste accumulation site is present, and that you...before starting the work. Before working with chemicals, first review their material safety data sheets...waste that may be contaminated with pathogens or chemicals that present a danger to people and the environment...disposal, commonly done with an autoclave. Only mechanical pipetting should be done in the laboratory. Don... evacuate and close off the area and alert the chemical hygiene officer. BSL-2 Guidelines Remember, the...

...nucleotides together. After ligation, the insert DNA is physically attached to the backbone and the complete plasmid...digestion . Most restriction enzymes digest DNA asymmetrically across their recognition sequence, which results...troubleshooting failed ligations. The following table indicates the various controls: Control Ligase Interpretation... treated vector Insert or water + Any colonies indicate contamination of intact plasmid in ligation or... 3:1 ratio is not working or when doing more complicated cloning. While 3:1 will get you in the ballpark...

...medium. This dot is composed of millions of genetically identical bacteria that arose from a single bacterium...plate to inoculate a bacterial culture for DNA purification will minimize the chance of having a mixture...draw in discontinuous lines. Start by streaking a vertical line of bacteria along one edge of the plate. ...

...Diagnostic Restriction Digest Introduction Sequence verification of important plasmid features (such as the gene...recommend 5’ and 3’ sequencing primers. These primers typically anneal to the backbone and can help you verify...sequencing results as a trace file (.ab1) which graphically depicts the sequence as a series of colored peaks...unreliable. Although your sequencing results may indicate bases at specific locations, by looking at the...

... . The pattern of the fragments on the gel can indicate if the plasmid contains the expected size insert...before use of more expensive forms of plasmid verification, such as DNA sequencing . In the example above...or enzymes) that cuts within the insert, but significantly off center (ideally around 1/3 of the way from...side of the insert, you can get a very obvious verification of the orientation so long as the expected products...

... these cells can still be used in downstream applications. Safety Warnings Lentivirus is generally considered...fluorescent cells Equipment Class II, Type A2 Biological Safety Cabinet 0.5–10 µL single channel pipette...tissue culture treated dish, Corning 3516 15 mL conical tubes, VWR 21008-216 Lentivirus preparation Reagent...You Start The health of the target cell line is critical for obtaining accurate titers. Check the cells...

...mounting medium Laboratory wipes 15 mL conical tubes 50 mL conical tubes Before Starting Refer to the manufacturer's...Guide Western Blot Protocol Recombinant Antibody Purification Protocol Introduction Immunocytochemistry is...as methanol or acetone may be better for some applications. Remove the paraformaldehyde and follow your...

...dispensed. The boxes that the tips come in often indicate a volume range that the tip can hold. This should... using, you will be rotating dials behind the numerical display or the top of the plunger of the pipette...no liquid should drip from the tip. This could indicate that the tip is not on the pipette properly. Place...pipette tip by holding the pipette tip over your dedicated waste container and pressing on the tip ejector...

... Agarose Gel Electrophoresis Agarose Gel DNA Purification Streaking and Isolating Bacteria Inoculating...instructions. If you want to perform plasmid purification without using a kit, you can find a protocol...Na-acetate (pH 4.8) Protocol: Generalized DNA Purification Grow an overnight culture of bacteria . Pro-...using. Store DNA at 4°C. Tips and FAQ Plasmid purification kits provide the fastest way to obtain a high...

... template region of DNA. They are synthesized chemically by joining nucleotides together. One must selectively...primers produce inaccurate, nonspecific DNA amplification product, and long primers result in a slower...which creates primer dimers and disrupts the amplification process. When designing, if unsure about what...

...Handling Visualization Signal Amplification Application Overview Quantification Methods Capture Methods Visualization...signaling molecules and are often used in clinical applications. Diabodies - Diabodies contain two Fab fragments...this can vary significantly based on the antibody and conjugate chosen. Signal Amplification The amount ...single assay. Overview of Antibody Applications Antibody-based applications can be generally classed into ...these applications, visit the Antibody section of the Addgene Protocols page. Antibody Applications - Quantification...processing steps, such as sonication. Denature proteins, using heat and/or chemicals, and run on an SDS-PAGE...concentrates in the sample. Controls for quantification methods Quantification methods should be run with: a positive...

...immunoprecipitation (enChIP) using CRISPR. Biochemical and Biophysical Research Communications . 439 (1), 132–136. PMID:... organisms, but modifications to various Cas enzymes have extended CRISPR applications to increasingly...takes place during the S and G2 phases. Chemically or genetically inhibiting genes involved in NHEJ or upregulating...genes. Each library typically contains ∼3–6 gRNAs per gene to ensure modification of every target gene...following the amplification protocol specified by the depositing lab. After amplification, you’ll need ...heritability. Not all modifications are stable across cell divisions, and some types of modifications are more frequently... subsequently isolated by affinity purification. After purification, molecules associated with the locus...

...Tetracycline 10 mg/mL 10 µg/mL Notes: Unless otherwise indicated, the antibiotic powder can be dissolved in dH ...test results. Sample Data In all cases below (-) indicates that the tested strain is not supposed to be resistant... resistant to the antibiotic, (+) indicates that the tested strain is supposed to be resistant to the ...