...gloves, and whenever possible, keep RNA sample and reagents cold and work quickly to reduce RNA degradation...degradation. Keep work area, equipment, and reagents RNase-free (Use an RNase decontamination solution, such as...Homogenizer Vortexer -20°C freezer -80°C freezer Reagents Solution D (for Protocol Option #1 ): 4 M Guanidinium...Sheet) for safety warnings and hazards for these reagents. Work in a well-ventilated space and under a fume... fume hood when working with the volatile reagents in the list above. Procedure Option #1 - Solution D...

...potential accidents such as spills. BSL-2 is different because it includes labs that work with agents associated...don’t touch your face! Close-toed shoes and long pants should be worn to protect your legs and feet from...wear glasses/goggles in addition to the BSL-1 requirements. Conclusion Although simple, following appropriate...

... and using a centrifuge to separate different components in a liquid sample....laboratory equipment used to separate different components in a liquid sample. It achieves this by using...appropriate for the biosafety level of the lab space Reagents Sample to be centrifuged Water Background Information... you use, most will have essentially the same components. The photo to the left shows a front view of ...

... and using a centrifuge to separate different components in a liquid sample Using a Light Microscope Learn...parts of a light microscope and its use Weighing Reagents Learn how to weigh laboratory materials on a balance...plasmid Gibson Assembly Combine overlapping DNA fragments in a single reaction Ligation Independent Cloning...Lentivirus Quantify virus using fluorescence measurements Colony Formation Titering Assay for Lentivirus...

...equipment, reagents, and methods that we use in our lab at Addgene. Equipment and reagents from other ...channel pipette 200–1000 µL single channel pipette Reagents 1X PBS Microcentrifuge tubes Sterile Poly-D-lysine..., incubation times, and recommended compatible reagents. Secondary antibodies must match the host species...

...therefore, the cells cannot be used for later experiments. Workflow Timeline Day 0: Seed and transduce ...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....titers as the likelihood of multiple integration events decreases as the dilution increases. Calculate ...

...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...