...that longer incubation times can reduce transfection efficiency. Add the OptiMEM + DNA + PEI solution to...handle a large number of T-175 flasks. Workflow Timeline Day 0: Seed cells in CS2 Day 2: Seed cells in ...in this protocol, but their conditions must be optimized. Plasmids for transfection: pHelper; pRC (Rep-...addition of acid or base. Add only a few drops at a time. Allow them to mix and recheck the pH to prevent... Close the bottle and mix by inverting multiple times Adjust pH to ~8.5 Filter sterilize through a 0.22...The health of the HEK293T cells is critical for optimal AAV yield. Do not overgrow your cells. Pass the...Complete. Pipet back and forth vigorously multiple times to obtain a single cell suspension (no clumps of...

... could lead to reduced transgene expression over time, as the lower expressing clones take over the polyclonal...individual cells with cloning cylinders. Workflow Timeline This protocol begins with a stable cell pool. ...theory a monoclonal line could be generated at any time. Considerations Before You Start Some cell lines...medium with increased serum concentrations, may be optimal for different cell types. Before starting this ... transgene expression. This protocol has been optimized for adherent cells but can be modified for suspension...because the buildup of waste products could be suboptimal for cell growth. This conditioned medium will...clumps into individual cells by passing several times through a serological pipet or by passing through...

...Update: February 13, 2019 Estimate of time required: ~3 h Workflow Timeline Plate set-up: 2 h qPCR run... very well by pipetting back and forth multiple times at each step Reagent Preparation Master Mix: Count...Keep track of the Ct value for each standard over time. They should remain within 0.5 Ct of their initial...the Ct value of the standard starts to drift, it’s time to make a new one. When developing the assay multiple... to pipet each dilution up and down at least 10 times, and use at least half of the final volume (mix ...mix well by pipetting back and forth at least 5 times. Seal plate with transparent film. Centrifuge at... U, Sing A, Ehrhardt A, Baiker A. Universal real-time PCR for the detection and quantification of adeno-associated...

...harvesting your cells. However, you should optimize the time based on your cell line and assay: Assay Days...Designing shRNA Oligos for pLKO.1 B.1 Determine the optimal 21-mer targets in your gene B.2 Order oligos compatible... F.1 Recommended materials F.2 Determining the optimal puromycin concentration F.3 Protocol for lentiviral...Designing shRNA Oligos for pLKO.1 B.1 Determining the Optimal 21-mer Targets in your Gene Selection of suitable...plasmid to obtain the ratio that gives you the optimal viral production. d. Create a master mix of FuGENE...located in the “Appendix”. F.2. Determining the Optimal Puromycin Concentration Each cell line responds...Addgene strongly recommends that you determine the optimal puromycin concentration for your cell line before...

...area and avoid breathing in the vapors. Workflow Timeline Day 1: Antigen Coating Day 2: Blocking Day 3: ...incubation and plate read Tips and Troubleshooting The optimal concentration of primary antibody to use will vary...999.5 mL 1X PBS Cap the bottle and invert several times to mix. Carefully remove the plate seal from the...to 25 mL of PBS. Cap the tube and invert several times to mix. Using a multichannel pipette, add 200 µL...into 50 mL PBS . Cap the tube and invert several times to mix. Dilute the primary antibody and an isotype...between 1–10 µg/mL. Cap and invert the tubes several times to mix. Using a multichannel pipette, add 100 µL...dilution buffer. Cap and invert the tube several times to mix. Using a multichannel pipette, add 100 µL...

... fixation method. The protocol may need to be optimized for different cells, target proteins, etc. Sharing...science. Last Update: January 20, 2022 Workflow Timeline Day 1: Seed cells Day 3-4: Fix and label cells...antibodies, such as antibody concentrations, incubation times, and recommended compatible reagents. Secondary ...concentration in antibody dilution buffer. Pro-Tip The optimal antibody concentration will vary but generally ...fluorescent filters. Tips and Troubleshooting The optimal fixation method will vary depending on the sample... find the best conditions for your target. The optimal antibody concentration will vary between antibodies...

...buffer and optimal antibody concentrations. Consider titrating your antibody to determine the optimal dose....approaches. Last Update: January 24, 2022 Workflow Timeline Day 1: Prepare lysates, run SDS-PAGE, transfer... concentration in blocking buffer. Pro-Tip The optimal concentration will vary between antibodies but ...detect the bands. Tips and Troubleshooting The optimal lysis buffer will vary depending on the sample ... buffers to find the best for your target. The optimal concentration and blocking buffer will vary between...consider titrating your antibody to determine the optimal dose. To ensure that your antibody is both functioning...

...regulations. Last Update: February 18, 2022 Workflow Timeline Day 1: Seed cells Day 2: Transfect cells Day 3...no more than 200 µL of NaOH or 20 µL of HCl at a time. Add deionized water to a final volume of 1 L and...resuspended before sampling. Gently swirl the flask 5–10 times before sampling. Transfer 10 µL of trypan blue into...microfuge tube containing the trypan blue. Pipette 10 times to mix. Load 10 µL of the cell suspension/trypan...the tube and vortex for 5 s to mix. Pro-Tip The optimal ratio of DNA:PEI may vary significantly and should...the flask dropwise. Cap the flasks and swirl 5–10 times to mix. Return the flask to the incubator. Section... Feed. Pro-Tip The feed can be repeated up to 4 times for a total of 16% of the culture volume. At 72-...

...reagents. Workflow Timeline Day 0: Seed HEK293T cells (or a subclone of HEK293T optimized for viral production...this protocol with a subclone of HEK293T cells optimized for viral production (AAVpro or Lenti-X), but ...addition of acid or base. Add only a few drops at a time, allow them to mix and recheck the pH to prevent...levels of virus. HEK293T cells should be split 3 times a week: Monday: Plate 1x10 6 cells in a 75 cm 2 ...are below passage 20 for viral production. The optimal mass DNA:mass PEI ratio will need to be empirically...

... cells, which come frozen and are prepared for optimal transformation efficiencies upon thawing. For the...flicking the bottom of the tube with your finger a few times. Pro-Tip Transformation efficiencies will be approximately... Pro-Tip This outgrowth step allows the bacteria time to generate the antibiotic resistance proteins encoded...plates at 37°C overnight. Tips and FAQ How can I save time when carrying out transformations? If you are not...grow up more of the plasmid) you can save a lot of time by shortening or skipping many steps and will still...your transformation procedure is working. TIP: Sometimes less is more. Although it may be counter-intuitive...

...ratios to optimize the ligation reaction. See Tips and FAQ below for details on optimization. Incubate...complementary, the two pieces of DNA connect and ultimately are fused by the ligation reaction. The example...is a good idea to take a fresh tube, thaw it one time and aliquot individual tubes of 5, 10 or 20μL for...plasmid in ligation or transformation reagents Optimizing the Vector:Insert Ratio Although a 3:1 insert...to vector ratio is usually sufficient, you can optimize the amount of insert and vector to improve ligation...

...be adapted to alternative cell lines. Workflow Timeline Day 0: Seed and transduce Cells Day 2–3 (am): ...cycles. Procedure Before beginning, determine the optimal dose of selective reagent for your target cell ...that they can grow out in a reasonable amount of time, but not so many that they vastly outnumber the ...important to do regular media changes and maintain optimal growth conditions for the surviving cells. Even... selection while the population expands. At this time, some researchers reduce the concentration of the...has the most desirable level of expression. Over time, transgene expression in a polyclonal population...

...However, if you want to store bacteria for a longer time, you will need to establish glycerol stocks. The...plasmid DNA prep the following day. Tips and FAQ The optimal concentration of long-term glycerol storage is ...glycerol. You can prepare the glycerol stock the same time you prepare your plasmid DNA. In the morning, when...not to freeze/thaw your glycerol stock too many times. Placing the glycerol stock on dry ice while streaking...that you shake the glycerol before freezing (5-6 times). Make sure that you see one uniform solution, and...

...regulations. Last Update: July 7, 2023 Workflow Timeline Day 1: Seed and transduce cells Day 4: Treat cells...When adding the virus to the diluent, pipette 10 times, to remove virus from pipette tip. To mix the dilution...set the P200 pipette to 200 µL and pipette 10–20 times. Use a new pipette tip to transfer 200 µL of the...seeding. Mix each well with a 1 mL pipette 5–10 times. The final volume in the well is 1.5 mL, and the...appropriate PCR tubes. Pipette back and forth 10 times to mix. Generating the Droplets Place a DG8 cartridge...following PCR parameters: Cycling Step Temperature (°C) Time (min) Ramp Rate (°C/sec) # Cycles Enzyme Activation...

... such as stable-cell line generation. Workflow Timeline Day 0: Seed 293T packaging cells Day 1 (pm): Transfect...addition of acid or base. Add only a few drops at a time, allow them to mix and recheck the pH to prevent...discard the tube and thaw a new working stock. The optimal mass DNA:mass PEI ratio will need to be empirically... high viral titer. 293T cells should be split 3 times a week: Monday: Plate 1×10 6 cells in a T75 flask... *Plasmid concentrations and ratios should be optimized for each transfer plasmid. Pro-Tip Endotoxins ...

...affinity purify recombinant antibodies. Workflow Timeline Day 1: Purify antibody Day 2 or later: Buffer ... discarding buffer from the collection tube each time. Place column in a new 50 mL LoBind collection tube...space in the column with PBS and pipette several times to mix. Centrifuge at 3100 x g for 8 min . Discard...container. Repeat steps 29-31 at least 4 additional times to ensure a full buffer exchange. Discard the flow...space in the column with PBS and pipette several times to mix. Centrifuge at 3100 x g for 8 min . Remove...

... in the 48-well dilution plate and pipette 5–10 times to mix. Dilute the virus as follows: Mix dilutions...generating aerosols. Be sure to change tips each time. Dilution 1 (20X): 5 µL in 95 µL 1X PCR buffer (...and mix by pipetting the liquid up and down 10–20 times. Gently cover the entire dilution plate with Microseal...appropriate PCR tubes. Pipette back and forth 5 times. Lightly cap the PCR tubes. Generate the Droplets...following PCR parameters. Cycling Step Temperature (°C) Time (min) Ramp Rate (°C/sec) # Cycles Denaturation 95...

...If so, you will likely need to grow for a longer time to get the correct density of bacteria since they...cell) and they need to grow for longer periods of time (approximately 18-30 hr). On the other hand, smaller...What went wrong? Try growing the culture for more time. Some bacterial cultures grow more slowly. Also,...rather than 37°C often require longer incubation times. Double check that the antibiotic in your LB media...

...double digest (digesting with two enzymes at the same time), you will need to determine the best buffer that...and the amount of DNA in the reaction, incubation time can range from 45 mins to overnight. For diagnostic... of methylation sensitive restriction sites . Sometimes enzymes cut sequences which are similar, but not...amount of Master Mix to each tube. This will save you time and ensure consistency across the reactions....

...the same time as the virus-mediated transduction. For some cell lines, transduction is optimized if the ...cannot be used for later experiments. Workflow Timeline Day 0: Seed and transduce cells Day 2: Replace...that case, polybrene should only be added at the time of viral transduction, and not during the cell seeding...