... a 1:100 dilution of the homogenized cell solution by adding 10 µL homogenized cell solution to 990 µL...Cloning by Limiting Dilution Isolating a Monoclonal Cell Population by Limiting Dilution You may also like...cell solution later in this protocol. Isolating a Monoclonal Cell Population by Limiting Dilution Isolate...homogenized cell solution is transferred. Transfer cells from the homogenized cell solution into the conditioned...cell solution at a concentration of 5 cells/mL. Prepare approximately 10 mL of this cell solution for ...medium, prepare this cell solution in fresh complete medium. This 5 cells/mL solution will be used to seed ... quantitate the homogenized cell solution. Homogenized cell solution concentration: 4 × 10 5 cells/mL ...

...in the dilution BSC. Prepare 1X dilution buffer (see recipe above). Save ~25 µL of 1X dilution buffer ... carefully add the dilution buffer to the dilution plate according to the dilution scheme listed in step...Pro-Tip Prepare the viral dilutions in a separate biological safety cabinet (dilution BSC) than that used for...touching the screen. Prepare the Serial Dilution Place a 48-well dilution plate in a chilled 96-well freezer... add 5 µL of each viral sample to Dilution 1 in the 48-well dilution plate and pipette 5–10 times to mix...change tips each time. Dilution 1 (20X): 5 µL in 95 µL 1X PCR buffer (1:20) Dilution 2 (20X): 5 µL in 95 ...PCR buffer (1:400) Dilution 3 (20X): 5 µL in 95 µL 1X PCR buffer (1:8,000) Dilution 4 (20X): 5 µL in 95...

...of 2 x 10 8 dilution 90 2 x 10 7 10 of 2 x 10 7 dilution 90 2 x 10 6 10 of 2 x 10 6 dilution 90 2 x 10 ...Total dilution Dilution 1 (DNase step) 5 uL AAV stock 45 uL 10X 10X Dilution 2 5 uL Dil. 1 95 uL 20X 200X...200X Dilution 3 20 uL Dil. 2 80 uL 5X 1000X Dilution 4 20 uL Dil. 3 80 uL 5X 5000X Dilution 5 20 uL Dil...25000X Dilution 6 20 uL Dil. 5 80 uL 5X 125000X Dilution 7 20 uL Dil. 6 80 uL 5X 625000X Dilution 8 20 ... To obtain a solution at 2 x 10 9 molecules/μL: 1.59 x 10 11 / 2 x 10 9 = 79.8X dilution ...10 5 10 of 2 x 10 5 dilution 90 2 x 10 4 10 of 2 x 10 4 dilution 90 2 x 10 3 Pro-Tip To help stabilize ... standard dilution. Dilute DNase-treated and AAV reference samples according to the dilution scheme in...

...transfer 200 µL of the first dilution into the next dilution. Repeat for all dilutions as indicated in the chart... virus dilutions were used to transduce cells: Initial dilution factor of virus stock Dilution factor ... µL 5 200 µL of 2.5-fold dilution 200 µL 400 µL 10 200 µL of 5-fold dilution 200 µL 400 µL 20 200 µL of... of 10-fold dilution 200 µL 400 µL 40 200 µL of 20-fold dilution 200 µL 400 µL 80 200 µL of 40-fold dilution... 160 200 µL of 80-fold dilution 200 µL 400 µL Add 150 µL of each viral dilution to a well of a 6-well ... viral dilution). Remember, this is an additional 10-fold dilution of the virus stock! Don’t forget to...~2 as you progress from the higher dilutions to the lower dilutions (blue). As shown in the image (Figure...

...EDTA Store Solution I at 4°C Solution II - Denaturing Solution 0.2 N NaOH 1.0% SDS Store Solution II at room...plasmid Resuspension buffer Denaturing solution Renaturing solution 2 mg/mL RNase A TE or water-saturated...that is compatible with the next solution. Add a denaturing solution to the resuspended bacteria. Note...contents, including plasmid DNA, into solution. Add a renaturing solution to the denatured bacteria. Note: ...Lysis Plasmid Miniprep Prepare the following solutions: Solution I - Resuspension Buffer 25 mM Tris-HCl (pH... room temperature Solution III - Renaturing Solution (Potassium Acetate) 120 mL 5M Potassium acetate 23...Resuspend the pellet in 100 μL of cold Solution I. Vortex the solution for 2 min or until all bacteria are...

... Mix the dilutions well Note: the 1:10 dilution can usually be omitted because this dilution typically... serial dilutions of the lentivirus into DMEM complete containing 10 μg/mL polybrene: Dilution Volume ...quantified. Add 150 μL of a viral dilution to each well (each well gets one dilution) Also include an untransduced... colonies for at least 2 of the dilutions. Pro-Tip The higher dilution wells tend to provide more accurate...present in the lower dilutions, and single colonies are visible in the higher dilutions. Last reviewed on...staining resistant colonies with crystal violet solution and, therefore, the cells cannot be used for later...DMEM complete containing 10 μg/mL polybrene by diluting 20 μL of 10 mg/mL polybrene into 20 mL of media...

...different incubation options available. Caution Use the Stop Solution in a well ventilated area and avoid ...Immediately before use, mix 5 mL TMB Solution and 5 mL Peroxide Solution from the TMB substrate kit. Cap the...determine the ideal concentration. Prepare serial dilutions of the purified antigen as follows: 2 ng/µL : ... wells of each row, using a different standard solution for each row of a 96-well ELISA microplate. Cover...the wash buffer from the wells. Prepare antibody dilution buffer (0.1% BSA in PBS) as follows: Dilute 50... to the desired concentrations in the antibody dilution buffer. Pro-Tip The ideal antibody concentration...multichannel pipette, add 100 µL of primary antibody solution to the appropriate wells. Cover the plate with...

...Option #1 - Solution D Protocol Before starting this protocol, make sure to prepare solution D (see reagent...or lyse tissues or cells in Solution D. For tissues: use 1 mL of Solution D per 100 mg of cells. For cultured...resuspension. This protocol provides two options: using Solution D (see reagent section for recipe) or using an...all-in-one acid guanidinium thiocyanate-phenol solution such as TRIzol®. For the precipitation step, two...reagents RNase-free (Use an RNase decontamination solution, such as RNaseZap® or RNase AWAY®, may be used... Vortexer -20°C freezer -80°C freezer Reagents Solution D (for Protocol Option #1 ): 4 M Guanidinium Thiocyanate...alcohol (49:1) 75% Ethanol RNase-free water or TE solution RNase free tubes: microcentrifuge tubes, 4 mL ...

...viral dilution to each well (each well gets one dilution, so a 6-well plate will hold 5 dilutions plus ...just a sample of possible dilutions. You may want to try higher/lower dilutions depending on your downstream...the results of your titration. Mix the dilutions well. Dilution Volume of Lentivirus (μL) Volume of DMEM...that already contain 0.5 mL of virus solutions at various dilution. Make sure to use the polybrene-containing...batch of DMEM complete + 10 µg/mL polybrene by diluting 20 µL of 10 mg/mL polybrene into 20 mL media. ...aliquot on ice prior to use. Prepare a range of dilutions of the lentivirus in DMEM complete + 10 µg/mL ...polybrene-containing media to make the cell solution in this step. To seed the cells: Prepare a batch of cells as ...

...swirling the flask occasionally as the solution heats up.). Caution HOT! Be careful stirring, eruptive boiling...do you get better resolution of bands? A few simple ways to increase the resolution (crispness) of your...gel electrophoresis, including tips to improve resolution and separation of bands. Protocols... completely dissolved (but do not overboil the solution, as some of the buffer will evaporate and thus...continue towards a boil. Keep an eye on it the solution has a tendancy to boil over. Placing saran wrap...necessary if you pay close attention. Let agarose solution cool down to about 50 °C (about when you can comfortably...0.2-0.5 μg/mL (usually about 2-3 μl of lab stock solution per 100 mL gel). EtBr binds to the DNA and allows...

...working solution: Prepare a 300 µM DAPI stock solution by diluting 2.1 µL of the 5 mg/mL DAPI solution to ... 300 nM DAPI working solution by diluting 5 µL of the 300 µM DAPI stock solution into 5 mL PBS. Protect... and 30 µL Triton X-100 in 10 mL PBS. Antibody dilution buffer: Dilute 0.5 g BSA and 150 µL Triton X-100...Remove the paraformaldehyde and follow your institution's laboratory safety guidelines for disposing of...antibody to the desired concentration in antibody dilution buffer. Pro-Tip The optimal antibody concentration...Counterstain nuclei with 500 µL of 300 nM DAPI working solution for 10 min at RT in the dark. Remove the DAPI ...

...hydrochloric acid until the solution clears. Check the pH of the solution and use hydrochloric acid or... 1 g/L glucose. 1 mg/mL polyethylenimine (PEI) solution: Dissolve 100 mg of PEI powder into 100 mL of ...to adjust the pH to 7.0. Pro-Tip The pH of this solution will drift pretty rapidly upon addition of acid...over or undershooting the desired pH. Allow the solution to mix for 10 min and then recheck the pH to ensure... ensure that it has not drifted. Filter the solution through a 0.22 μm membrane. Aliquot 500–1000 μL into... Store the tubes at -80 °C. After thawing, the solution can be stored at 4 °C for up to 2 months. After...working stock. 40% POLYETHYLENE GLYCOL (PEG) 8000 solution: Dissolve 400 g of PEG 8000 and 24 g of NaCl ...

...will be higher titer, consider additional dilutions. Dilution Volume of Lentivirus Stock (μL) Volume of... Mix the dilutions well Gently aspirate media from the cells. Add 1.5 mL of a viral dilution to each well...multiple dilutions. Sample Data Figure 1: 293T cells were transduced with a range of dilutions of pHAGE-TO-dCas9...ensure that you are in compliance with your institution’s biosafety regulations. Workflow Timeline Day...lentiviral aliquot on ice prior to use. Prepare dilutions of the lentivirus into DMEM complete containing... well (each well gets one dilution with one well remaining as the untransduced control). Count the cells...transduction units per mL (TU/mL) using either the dilution factors (method 1) or the volume of virus (method...

...ensure that you are in compliance with your institution’s biosafety regulations. Last Update: February...Leupeptin, Millipore Sigma L2884 Aprotinin saline solution, Millipore Sigma A6279 Before Starting Warm the...µm filter. Prepare 10 mL aliquots and store the solution at -20 °C. 1 mg/mL PEI-MAX Add 1 g of PEI-MAX ...but check for the presence of particles still in solution. Continue to stir until all particles have dissolved... to ensure that it has not drifted. Filter the solution through a 0.22 µm filter. Prepare aliquots and...at 4 °C until use. We suggest preparing fresh solutions after one month. BCD Feed 500 mL BalanCD HEK293...at 4 °C until use. We suggest preparing fresh solutions after one month. 1000X protease inhibitor cocktail...

...must contact your institution’s Bio-Safety office to receive permission and institution-specific instructions...sulfate for polybrene. c. Add lentiviral particle solution from step E. For a 6 cm target plate, add between...Please follow all safety guidelines from your institution and from the CDC and NIH for work in a BL2 facility...safety practice to follow, please contact your institution’s safety office. For more information, please ...extract 10g sodium chloride 15g agar Prepare LB agar solution by dissolving 40g of LB powder in 1L of distilled...Hexadimethrine Bromide (Polybrene) Prepare a 1mg/mL solution of polybrene (Sigma-Aldrich catalog #H9268) in...in 0.9% NaCl. Autoclave to sterilize. Stock solution is stable at 4°C for up to one year. The powder form...

...cuvette. Pulse. Caution Electroporation involves the use of high voltages, please use caution when activating... a second and then third 1:100 dilution). Plate 100 µL of each dilution onto a prewarmed Petri dish. Incubate...adequate time to fully gel and to dry slightly. We routinely use 350 mL of LB Agar per bioassay plate. 14 mL... mix the four tubes. Perform sequential 1:100 dilutions of the cells (add 10 µL of the pool to 990 µL ... colonies can be too great to count. Ideally, dilutions would have been sufficient to enumerate single...plate, count the second plate (ie. the 1:10,000 dilution). After 12-18 hours of growth, use spreader and...suspended in cryoprotectant like Glycerol or DMSO solutions. Quantify the individual Maxipreps by Nanodrop...

...hydrochloric acid until the solution clears. Check the pH of the solution Use hydrochloric acid or sodium...hydroxide to adjust the pH to 7.0. Typically, the solution will be basic and will need adjustment with hydrochloric...hydrochloric acid first. Pro-Tip The pH of this solution will drift pretty rapidly upon addition of acid...over or undershooting the desired pH. Allow the solution to mix for 10 min and then recheck the pH to ensure... ensure that it has not drifted. Filter the solution through a 0.22 µm membrane. Aliquot 500–1000 µL into.... Store the tubes at -80 °C. After thawing the solution can be stored at 4 °C for up to 2 months. After...

...hydrochloric acid until the solution clears. Check the pH of the solution. Use hydrochloric acid or sodium...hydroxide to adjust the pH to 7.0. Typically, the solution will be basic and will need adjustment with hydrochloric...hydrochloric acid first. Pro-Tip The pH of this solution will drift rapidly upon addition of acid or base...over or undershooting the desired pH. Allow the solution to mix for 10 min and then recheck the pH to ensure... ensure that it has not drifted. Filter the solution through a 0.22 μm membrane. Aliquot 500–1000 μL into... tubes at -20 °C or -80 °C. After thawing, the solution can be stored at 4 °C for up to 2 months. After...

...preparations contain ~20% empty particles. 60% iodixanol solution is available under the name OptiPrep (Link opens...iodixanol and 45 μL of phenol red Underlay each solution into a QuickSeal tube in the order below using...the tube is punctured, AAV-containing Iodixanol solution will flow from the needle set at the 40–60% interface...microcentrifuge tubes are well positioned to collect the solution, or you will lose a significant amount of your...exchange: Stocks of Pluronic F68 Pluronic F68, 10% solution A: 0.1% in PBS: 49.5 mL PBS + 500 µl Pluronic .... Figure 3: Example of an SDS-PAGE gel of 10 consecutive gradient fractions followed by silver stain. ...

...should be properly disposed of following your institution’s hazardous waste procedures. Last Update: February...Millipore Sigma S7907-500G 1 M Trizma hydrochloride solution pH 9.0, Millipore Sigma T2819-1L 250 mL sterile...BD302832 Filter, 0.2 µm (luer-lock), VWR 431229 IgG Elution Buffer, Thermo Fisher 21028 Protein G GraviTrap... Warm the affinity column, binding buffer, and elution buffer to room temperature (RT) before use. Reagent... columns at the bottom. Add 5 mL of Pierce IgG Elution Buffer to the capped Gravitrap column and incubate...column at 1000 x g for 2 min to remove storage solution. Place a mark on the side of the column where ...