...equipment, reagents, and methods that we use in our lab at Addgene. Equipment and reagents from other ...bath Vortex Stir bar Magnetic stir plate pH meter Reagents HEK293 cells Recombinant antibody plasmid DNA ...Pluronic-F68 40 mL 200 mM Glutagro Do not add selective reagents Store at 4 °C until use. We suggest preparing ...

...protein expression studies. Moreover, repeating experiments in a stable cell line, as opposed to transiently-transfected...incubator Pipet controller Hazardous waste container Reagents DMEM high glucose, Corning 10-013-CV L-alanyl-...lines. It is not recommended that lentiviral supernatants be subjected to multiple freeze-thaw cycles....may also exhibit varying numbers of integration events. In other words, if an MOI >1 was used, some cells...

...equipment, reagents, and methods that we use in our lab at Addgene. Equipment and reagents from other ...optional) Scale Microsoft Excel or similar software Reagents Purified antigen Purified recombinant antibody...Tween-20, VWR 0777-1L Foil Before Starting Warm reagents to room temperature. Procedure Section 1: Prepare...

...population that varies in the number of integration events and the site(s) of proviral integration across ...Microscope Hemocytometer or other cell counter Reagents Polyclonal stable cell pool DMEM high glucose,...using selective medium throughout subsequent experiments. When selective medium is used, it may have additional... that the use of selective medium throughout experiments be determined empirically, and that the cell ...

..., where TU is transducing units and generally represents the number of infectious viral particles. Users... Mini Centrifuge, Thermo Scientific, 10199-452 Reagents GeneJet Genomic DNA Purification Kit, Thermo Fisher...equipment with 10% bleach prior to use and keeping all reagents and samples on ice or pre-chilled 96-well freezer...during use. To reduce the risk of contaminating reagents we recommend making small aliquots of master mixes...

...Disinfectant Thermometer Water bath weights and floats Reagents None needed Procedure Ensure that the water bath...prevent growth of bacteria or fungi. There are disinfectants designed to be used in water baths with instructions...clean out the water bath even if you are using disinfectants as described above in step 3. You will also ...

...overhangs to form a stable association between fragments, allowing for transformation without ligation....Vector In this example, the vector is cut at two points by the enzyme BsaI, removing the stuffer sequence... A typical T4 Pol reaction is shown. Mix the components (add the T4 last) and incubate at room temperature...

...purification allows you to isolate and purify DNA fragments based on size. Following gel electrophoresis, ...and Australia, and advice for current graduate students considering their future careers. Eric J. Perkins...Business Team, his hobbies, and advice for grauated students pursuing non-academic careers....

...waste container Inverted fluorescence microscope Reagents DMEM high glucose, Corning 10-013-CV L-alanyl-... It is not recommended to subject lentiviral supernatants to multiple freeze-thaw cycles. Procedure Seed...you risk counting cells with multiple integration events leading to underestimation of the true titer. Calculate...

...Pipetman Reagents Liquid DNA aliquot of your plasmid of interest (see below for recommend amounts) Appropriate...Master Mix" consisting of all of the reaction components except for the DNA. Aliquot your DNA into individual...

...works. Equipment Autoclave Water bath Pipetman Reagents 37 g pre-mixed powder consisting of: 5.0 g yeast...bottle with your initials, the date, and the bottle contents. This will clear up any confusion later if your...recommended temperature should kill most potential contaminants, certain types of spores will stubbornly survive...

...equipment, reagents, and methods that we use in our lab at Addgene. Equipment and reagents from other ... Gel imaging system ImageJ or similar software Reagents Purified recombinant antibody, 0.9–1.1 mg/mL SimplyBlue...

...equipment, reagents, and methods that we use in our lab at Addgene. Equipment and reagents from other ...channel pipette 2–20 µL single channel pipette Reagents Aspirating pipette, VWR 414004-265 5 mL pipette...

...equipment, reagents, and methods that we use in our lab at Addgene. Equipment and reagents from other ...Platform shaker Cold room Gel imager -80 °C freezer Reagents 1X PBS Lysis buffer e.g., RIPA lysis buffer Microcentrifuge...

...Magnetic stir bar Sonicator Ear protection Vortex Reagents Adherent HEK293T cells (ideally AAV-293T clones...Polyethylenimine (PEI) 25 kDa MW Pro-Tip Other transfection reagents may be used in this protocol, but their conditions... with PEI and allow for the addition of other components, such as sorbitol and sodium bicarbonate, sorbitol...

...(or other small flame source) Incubator Marker Reagents LB agar plate (with appropriate antibiotic) Bacterial...DNA or use the individual colonies for other experiments....

...burner (or other small flame source) Incubator Reagents 6 cm diameter LB/agar plates without antibiotic...will work for this assay, and the above result represents a single experiment. For publishable data, the...

...purification allows you to isolate and purify DNA fragments based on size. The procedure starts with standard...Gel Electrophoresis Protocol with the following amendments: Notes: Gel purification is most efficient with...

...different cell lines and different transfection reagents. Workflow Timeline Day 0: Seed HEK293T cells (...incubator Pipet controller Hazardous waste container Reagents DMEM high glucose, Corning 10-013-CV L-alanyl-...

...alternative packaging cell lines or transfection reagents. Once produced, lentivirus can be used for a variety...container pH meter Stir plate Magnetic Stir Bar Reagents DMEM high glucose, Corning 10-013-CV L-alanyl-...