...counter Reagents Polyclonal stable cell pool DMEM high glucose, Corning 10-013-CV L-alanyl-L-glutamine ...alternative, such as glutaGRO) To a 500 mL bottle of DMEM high glucose, add 55 mL of heat-inactivated FBS and 5...for one 96-well plate. Allow the cells to grow overnight. Replace the medium on the cells and collect the...conditioned medium, it will be more accurate if a higher volume of homogenized cell solution is transferred... Western blotting to screen for lines with the highest or lowest transgene expression ( Figure 2 ). Sample...

...PES. Using a PES filter will maximize titer . DMEM high glucose, Corning 10-013-CV DMEM, low glucose (1 ...alternative, such as glutaGRO) To a 500 mL bottle of DMEM high glucose, add 55 mL of heat-inactivated FBS and 5...dissolved. Pro-Tip This step is challenging due to the high viscosity of PEG. Stirring under medium heat will...precipitation. Precipitation of the viruses can proceed overnight at 4 °C if needed. Transfer the entire sample ... prep . The clarified supernatant can be kept overnight at 4 °C before proceeding with the purification...

...Inoculating an Overnight Liquid Culture . After you have bacterial growth, add 500 μL of the overnight culture...bacteria onto an LB agar plate . Grow your bacteria overnight at the appropriate temperature. Growth conditions...page. The next day you will be able to start an overnight culture for plasmid DNA prep the following day...

...container Inverted fluorescence microscope Reagents DMEM high glucose, Corning 10-013-CV L-alanyl-L-glutamine ...alternative, such as glutaGRO) To a 500 mL bottle of DMEM high glucose, add 55 mL of heat-inactivated FBS and 5...each well of the 6-well dish. Incubate the cells overnight. If using freshly collected virus, filter through...If you anticipate that your viral stock will be higher titer, consider additional dilutions. Dilution ...

...protocol may need to be adjusted to accommodate slight differences between products. Addgene does not ...mg/mL DAPI solution to 100 µL PBS. Protect from light. Prepare a 300 nM DAPI working solution by diluting...DAPI stock solution into 5 mL PBS. Protect from light. Procedure Section 1: Seeding cells Place a sterile...Pro-Tip The plate can be wrapped in foil to block light. Remove the secondary antibody and dispose of it...

...controller Hazardous waste container Reagents DMEM high glucose, Corning 10-013-CV L-alanyl-L-glutamine ...alternative, such as glutaGRO) To a 500 mL bottle of DMEM high glucose, add 55 mL of heat inactivated FBS and 5...colonies for at least 2 of the dilutions. Pro-Tip The higher dilution wells tend to provide more accurate titers...dilutions, and single colonies are visible in the higher dilutions. Last reviewed on: August 31, 2023...

...to begin using the pipette. To the right is a photo of how you might hold a pipette. Without moving past... P1000 pipette shows the volume adjustment ring right below the plunger. This ring changes the pipette...P20, P100, P200, and P1000 as shown from left to right. Pipette Dispense Volume P2 0.2 to 2 µL P10 1 to...

...purified AAV. Option #3 Puncture the QuickSeal tube slightly below the 60–40% interface with an 18 ga needle... (1999): 973-985 (Link opens in a new window) . Right panel: cartoon indicating the position of the needle...harvesting of the purified AAV using option #1. Right image adapted by permission from Macmillan Publishers... viral vectors," (Link opens in a new window) copyright (2006). Figure 3: Example of an SDS-PAGE gel of...

...viral particles. Users may need to run lower or higher dilutions depending on their particular sample....tissue culture treated dish, VWR, 29442-042 DMEM, high glucose, pyruvate, Corning, 10-013-CV Heat-inactivated... Reagent Preparation DMEM Complete: 500 mL DMEM, high glucose, pyruvate 55 mL heat inactivated premium...increases by a factor of ~2 as you progress from the higher dilutions to the lower dilutions (blue). As shown...

...spectrophotometer uses the absorbance/transmission of light through a liquid to determine the concentration ...liquid. Molecules absorb different wavelengths of light to varying degrees and most have a specific wavelength... absorbance, you need to know the wavelength of light that your substance maximally absorbs. In the case...

...protocol may need to be adjusted to accommodate slight differences between products. Addgene does not ...platform. Pour off the water in the sink. Take a brightfield image of the gel with an appropriate imaging ...25 kDa corresponding to the heavy chain (HC) and light chain (LC) proteins, respectively. There should ...

...protocol may need to be adjusted to accommodate slight differences between products. Addgene does not ...through a 0.2 µm PES filter. Aliquot and freeze upright at -20 °C Procedure Section 1: Affinity chromatography...and gently pour off the ethanol storage buffer. Tightly attach LabMate PD10 Buffer Reservoirs to the top...

...bases. This is an example of a trace file from a high-quality portion of a sequencing reaction: Sequence...file from a portion of a sequencing reaction with high background: If you obtain a different sequence than...

...Distilled water Disinfectant Thermometer Water bath weights and floats Reagents None needed Procedure Ensure...be used to secure tubes in place and water bath weights can hold bottles in place. After putting your tubes...

...extinguisher Guidelines BSL-1 Guidelines Before You Work Right after entering the lab, put on the proper personal.... BSL-2 centrifugation steps require an aerosol-tight lid and rotors should be loaded and unloaded in ...

...reaction, incubation time can range from 45 mins to overnight. For diagnostic digests, 1-2 hours is often sufficient...and can happen for a variety of reasons, including high glycerol concentration. Learn more at (Link opens...

...Incubate at room temperature for 2hr, or at 16°C overnight (following the manufacturer’s instructions). Note...For many ligation reactions, especially if using "high concentration" ligase, 5min at room temperature ...

...your plasmid and order primers (see figure to the right). When designing your plasmid, think about what ...fragments can subsequently anneal to each other. Phusion High-Fidelity DNA Polymerase - incorporates nucleotides...

...protocol may need to be adjusted to accommodate slight differences between products. Addgene does not ... 0.2 µm PES syringe filter. Aliquot and freeze upright at -20 °C. Procedure Section 1: Seeding cells The...

...viral particles. Users may need to run lower or higher dilutions depending on their particular sample....appropriate PCR tubes. Pipette back and forth 5 times. Lightly cap the PCR tubes. Generate the Droplets Bring ...