...2022 Workflow Timeline Day 1: Seed cells Day 2: Transfect cells Day 3-6: Feed cells Day 7: Harvest antibody...fresh solutions after one month. BCD Feed 500 mL BalanCD HEK293 Feed 20 mL 200 mM Glutagro Store at 4 °C... °C. Procedure Section 1: Seeding cells The day prior to transfecting, seed a 108 mL culture of HEK293...post-transfection, supplement the flask with 4% BCD Feed. Pro-Tip The feed can be repeated up to 4 times for a total...Example feeding schedule: Thursday: Transfect cells. Friday (24 h post-transfection): Add 4% BCD Feed. Monday...to 3.75 mM, add 4% BCD Feed. Tuesday (120 h post-transfection): Add 4% BCD Feed. Wednesday (144 h post-transfection...purified for use in a variety of applications. Sharing speeds science. We believe that sharing the full details...

...control panel where you can set the time and speed needed for your experiment. Procedure An example of...centrifuge needed. This decision should be based on the size of tubes you are using and the speed at which...according to speed and time (and temperature, if applicable) listed in your protocol. Pro-Tip Spin speed is often...which is generated by spinning the sample at a high speed. Being able to separate solids from a liquid or ...accommodate different sized containers, spin at different speeds, or keep samples at specific temperatures. This...conical tubes. Depending on the centrifuge their speed ranges can vary, but are typically lower than some... about bench height. They often spin at similar speeds to their smaller cousins, but can hold much larger...

...QuickSeal tube spacers 16 ga needle 18 ga needle 10 mL syringe 18 ga blunt edge needles, Hamilton 1X PBS pH 7.4...Figure 2) with an 18 ga needle. Place the first microcentrifuge tube under the needle’s opening to collect ...interface with an 18 ga needle attached to a 10 mL syringe. The bevel of the needle should be up, facing ...a 10 mL syringe and a blunt edge 18 ga Hamilton needle, taking care to avoid bubbles (Figure 1). 8 mL ... 90 min in a T70i rotor at 10 °C. Pro-Tip If you need more time, you can alternatively centrifuge for ...Puncture the top of the QuickSeal tube with a 16 ga needle and start collecting 0.5 mL to 1 mL fractions per...AAV-containing Iodixanol solution will flow from the needle set at the 40–60% interface. Make sure that the...

...175 flasks. Workflow Timeline Day 0: Seed cells in CS2 Day 2: Seed cells in CS5 Day 3 (am): Transfect cells... for 24 h–36 h. Proceed with transfection: Calculate the amount of each plasmid needed to have a 1:1:1...plasmid we need: Sample Calculations RepCap: 0.08 μg/bp × 7,265 bp = 582.1 μg Volume Needed: 582.1 μg ...Precipitation of the viruses can proceed overnight at 4 °C if needed. Transfer the entire sample to 3 ...volume to 300 mL with DMEM complete media and mix. Seed all cells in 1 CS2. Return to incubator for 48 h...Count cells using a hemocytometer or cell counter. Seed 350 million cells from the CS2 into one CS5 with...three plasmids, we can determine the total μg/bp we need to achieve a 1:1:1 molar ratio of each plasmid: ...

...optional) Seed cells for the generation of conditioned medium (see below for more details) Day 1: Seed individual...mL To seed one 96-well plate, make 10 mL of a 5 cell/mL solution. Calculate the total cells needed: Total...doing this, you are seeding the plate at an average density of 0.5 cells/well. Seeding an average of 0.5...Procedure Generating Conditioned Medium (optional) Seed stable cells such that the next day they will be...cells in a 10 cm dish. Each 10 cm dish should be seeded in 10 mL DMEM complete, which will generate enough...cell solution for each 96-well plate you plan to seed. If you choose not to use conditioned medium, prepare...medium. This 5 cells/mL solution will be used to seed the 96-well plate. Sample Calculation First, use...

... chemical reactions such as restriction digests needed in molecular biology, and thaw cell lines. There...Thermometer Water bath weights and floats Reagents None needed Procedure Ensure that the water bath and water ...clean it regularly. Determine the amount of water needed to fill the water bath, and be sure to know what...will reach the desired temperature by the time you need it for your experiment. Water baths tend to heat...up the water bath 30 minutes to 1 hour before you need to use it to allow the temperature to stabilize....containers that will go into the water bath. If you’ll need to sterilize your items after removing them from...tube. This way, as items are floating, you do not need to necessarily maneuver the bottles or tubes to ...

...restriction site to the reverse primer. Next, we need to examine the DNA sequence that we want to amplify...the Reverse Primer, the design is similar, but we need to use the reverse complement to get PCR amplification...(30bp with 18bp of homology to the ORF). We now need to generate the reverse-complement of this sequence...wide range of annealing temperatures, but you may need to increase your primer length and increase the ...overhangs or no overhangs after digestion, you will need to use a phosphatase to prevent re-circularization...self-ligating recipient plasmid backbone. Transformation Proceed with the transformation according to the manufacturer.... Isolate the Finished Plasmid Finally, you will need to pick individual bacterial colonies and check ...

...against the target to show a dose response. Sharing speeds science. We believe that sharing the full details.... However, please be aware that the protocol may need to be adjusted to accommodate slight differences...concentration of primary antibody to use will vary and needs to be empirically determined. If possible, run an... in a microfuge tube and vortex. Pro-Tip You may need to try a few different concentrations of antigen... The ideal antibody concentration will vary and needs to be empirically determined. We suggest starting...product indicates the presence of too much HRP and the need to optimize experimental conditions. When the desired...the standard curve will vary between targets and needs to be empirically determined. The dilution series...

...protocol may need to be optimized for different cells, target proteins, etc. Sharing speeds science. We.... However, please be aware that the protocol may need to be adjusted to accommodate slight differences...Update: January 20, 2022 Workflow Timeline Day 1: Seed cells Day 3-4: Fix and label cells Equipment Pipette... PBS. Protect from light. Procedure Section 1: Seeding cells Place a sterile poly-D-lysine coated coverslip...each well of a 24-well cell culture treated plate. Seed 5 x 10 3 HeLa cells per well. Allow the HeLa cells...sample type and the protein of interest. You may need to try a variety of fixation methods to find the...

... for later experiments. Workflow Timeline Day 0: Seed and transduce cells Day 2: Replace media with fresh...containing selection reagent Days 3–14: Change media as needed Days 14–18: Stain cells and count resistant colonies...freeze-thaw cycles. This protocol outlines the seeding of the cells at the same time as the virus-mediated...lines, transduction is optimized if the cells are seeded the day before transduction. Note that in that ...of viral transduction, and not during the cell seeding step. Procedure Before beginning a colony formation...antibiotic required to kill your target cell line needs to be empirically determined. Treat the target cells...well by using 150 μL of media in place of virus. Seed 1,000 cells into each well of a 6-well dish. Prepare...

...the lab! Introduction For many experiments, you’ll need to make buffers, media, or other solutions. A key...out your reagents, determine the amount that you need to weigh. Gather the reagent you will be weighing...put the reagent in. For some reagents, you might need to weigh it out in the fume hood to prevent exposure...you’re weighing out is within this range. If you need less than a gram of material, use an analytical ...out the reagents in batches. For example, if you need 300 g of sucrose, you can weigh out 100 g three ...the surface. Tare the weighing boat or paper. You need to do this because you don’t want to include the...

... batch needs to be validated and the best ratio of mass DNA:mass PEI determined. Procedure Seed HEK293T... transfection reagents. Workflow Timeline Day 0: Seed HEK293T cells (or a subclone of HEK293T optimized.... Typically, the solution will be basic and will need adjustment with hydrochloric acid first. Pro-Tip...production. The optimal mass DNA:mass PEI ratio will need to be empirically determined for each new batch ... 10 cm plate Pro-Tip The ratio of µg DNA:µg PEI needs to be empirically determined. Once a batch of PEI...

...Petri dish (VWR, 11019-552) 4 MaxiPreps (Qiagen HiSpeed Max, Catalog #12663) Tips (1000 µL, 200 µL, 10 ...side up until dried before overnight incubation if needed. Place 100 mL sterile LB at 4 ℃. Day 2 (morning...one to two more conical tubes on ice in case you need to spread out the harvested cells further than four...bubbles or pour off plates into conical tubes as needed. Add 10 mL cold LB to each plate for each scrape... Maxiprep. Purify plasmid DNA using the Qiagen HiSpeed Maxi Kit (one conical is its own Maxiprep). Critical...reagents (not including the recovery media!). Do not proceed with Maxipreps or NGS until adequate transformation..., scale the reagent volume and column number as needed. The use of Mega or Gigapreps is acceptable when...

...stable-cell line generation. Workflow Timeline Day 0: Seed 293T packaging cells Day 1 (pm): Transfect packaging.... Typically, the solution will be basic and will need adjustment with hydrochloric acid first. Pro-Tip... stock. The optimal mass DNA:mass PEI ratio will need to be empirically determined for each new batch ...below passage 15 for viral production. Procedure Seed 293T packaging cells at 3.8×10 6 cells per plate...working stock, therefore the ratio of μg DNA:μg PEI needs to be empirically determined. Once a batch of PEI...Gently aspirate the media out of the previously seeded 10 cm plate. Slowly pipette the transfection mix...

...your gene of interest (YGOI for short). You might need to express YGOI in cultured mammalian cells. The... the recipient plasmid's MCS. However, you still need to avoid restriction enzymes that cut within your...overhangs or no overhangs after digestion, you will need to use a phosphatase to prevent re-circularization.... Isolate the Finished Plasmid Finally, you will need to pick individual bacterial colonies and check ...(the more background, the more colonies you will need to pick) and grow overnight cultures for DNA purification... used enzymes with compatible overhangs you will need to verify the orientation of your insert, so you...

...supernatant using Protein A or Protein G columns. Sharing speeds science. We believe that sharing the full details.... However, please be aware that the protocol may need to be adjusted to accommodate slight differences...have a finite binding capacity. If your sample exceeds the capacity, divide the sample among multiple ...concentration of the pooled sample is above 1.0 mg/mL proceed to Option 1 with a buffer exchange using a Zeba...concentration of the pooled sample is below 1.0 mg/mL proceed to Option 2 with a buffer exchange/concentration...Spectrophotometer. Dilute antibody to 1 mg/mL with PBS if needed. For long term storage, add sterile sodium azide...

... are necessary when running a PCR reaction. One needs to design primers that are complementary to the ...complementary to template strand). However, primers do not need to correspond to the template strand completely;...completely to the template DNA strand so elongation can proceed. Usually a guanine or cytosine is used at the 3...hybridizing rate. On average, the DNA fragment that needs to be amplified should be within 1-10 kB in size...secondary structure to avoid internal folding. One also needs to avoid primer-primer annealing which creates primer...

...strains require growth at 30°C. If so, you will likely need to grow for a longer time to get the correct density...OD600 to measure the density of your culture if needed. A good negative control is LB media + antibiotic...For long term storage of the bacteria, you can proceed with Creating a Glycerol Stock . You can now isolate...approximately one or two copies per cell) and they need to grow for longer periods of time (approximately...copy number. High copy number plasmids should only need to be grown for 12-16 hr on average. Certain features...

... number of infectious viral particles. Users may need to run lower or higher dilutions depending on their...Last Update: July 7, 2023 Workflow Timeline Day 1: Seed and transduce cells Day 4: Treat cells with Benzonase...calculations later (see calculation example below). Seed 300,000 cells/well in 1350 µL media and 11.1 µg/...and the untransduced control. Pro-Tip For even seeding, prepare a batch for 10 wells with 3,000,000 cells...Polybrene. Mix the cell suspension well before seeding. Mix each well with a 1 mL pipette 5–10 times. ...Seal’ button. After the plate has been sealed, proceed to thermocycling. Thermal Cycling Run the following...

...digesting with two enzymes at the same time), you will need to determine the best buffer that works for both...-0.5 µL will likely be more enzyme than you will need, but that's okay because a little more enzyme is...buffer), but will not be gel purifying it, you may need to inactivate the enzyme(s) following the digestion...you cannot find compatible sticky ends, you will need to fill in the overhangs and conduct a blunt end...ends or a single enzyme to cut the vector, you will need to use a phosphatase to prevent re-circularization...