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...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...

... 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...

... 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 h on average. Certain features...

...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...

... as when you have a tube of plasmid DNA and just need to transform bacteria so that you can grow up more...the transformation, so when higher efficiency is needed follow the complete protocol. Thaw the competent...less efficient at taking up larger plasmids. If you need to transform large plasmids, it is a good idea to...induce membrane permeability. To do this you will need to have access to an electroporator and the appropriate...

...concentrations of 0.7% to 2% depending on the size of bands needed to be separated - see FAQs below . Simply adjust...add EtBr to the gel and running buffer, you will need to soak the gel in EtBr solution and then rinse ... procedures, such as molecular cloning, you will need to purify the DNA away from the agarose gel. For...want to load on a gel, make 10% more volume than needed because several microliters can be lost in pipetting...

...alternative cell lines. Workflow Timeline Day 0: Seed and transduce Cells Day 2–3 (am): Remove media, ...containing selection reagent Day 3–14: Change media as needed Day 14–18: Expand and harvest stable cell lines...control well). Perform a "reverse transduction" by seeding 50,000 cells into each well of the 6-well dish....media to make the cell solution in this step. To seed the cells: Prepare a batch of cells as follows: ...

...precast SDS-PAGE gel, and immunoblotting. 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...protein is in low abundance in the sample you will need to load a greater amount of total protein. Prepare...cellular location of the protein of interest. You may need to try a variety of lysis buffers to find the best...

...healthy humans. BSL-2 includes all of the precautions needed in BSL-1, however there are additional precautions...shoes Gloves Eye protection and face shields, as needed Guidelines Your lab coat should be buttoned to ...such as goggles and/or face shields can be used as needed. For BSL-2 work always wear glasses/goggles in ...

...a glycerol stock or stab culture of bacteria and need to purify plasmid DNA from it, you will want to ...hours) at 37 °C. Pro-Tip Some plasmids or bacteria need to be grown at 30 °C instead of 37 °C. This is ...colonies. Once you have single colonies, you can proceed to Recovering Plasmid DNA or use the individual...