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We narrowed to 50 results for: URE

Showing: 21 - 40 of 50 results

  1. Protocol - How to Create a Bacterial Glycerol Stock

    Type
    Protocol
    ...glycerol stocks. Procedure Follow the steps for Inoculating an Overnight Liquid Culture . After you have... appropriate temperature. Growth conditions, including copy number and growth temperature, can be found...you retrieve your liquid bacterial culture, take 500 μL of culture to make your glycerol stock before ...times). Make sure that you see one uniform solution, and there are no layers present. Be sure to label both...Transformation Recovering Plasmid DNA from Bacterial Culture Background Information Bacterial glycerol stocks... bacterial growth, add 500 μL of the overnight culture to 500 μL of 50% glycerol in a 2 mL screw top tube...next day you will be able to start an overnight culture for plasmid DNA prep the following day. Tips and...

  2. Lentivirus Production

    Type
    Protocol
    ...cm tissue culture plates. Incubate the cells at 37 °C, 5% CO 2 for ~20 h. Prepare a mixture of the three... 113.4 Gently add the diluted PEI mixture to the diluted DNA mixture. Add the diluted PEI dropwise while...polyethylenimine (PEI) transfection protocol. This procedure can be modified for alternative packaging cell...Microcentrifuge tubes, Neptune 3745.X 10 cm tissue culture dish, Corning 430167 15 mL conical tubes, VWR 21008...solution to mix for 10 min and then recheck the pH to ensure that it has not drifted. Filter the solution through...that are below passage 15 for viral production. Procedure Seed 293T packaging cells at 3.8×10 6 cells per...negative E. coli strain such as NEB stable. Make a mixture of a total of 500 μL PEI-OptiPro SFM with enough...

  3. Transfection for Recombinant Antibodies

    Type
    Protocol
    ... counter and measure the live cell density and viability of the culture. Pro-Tip Culture should be between...20 °C. Procedure Section 1: Seeding cells The day prior to transfecting, seed a 108 mL culture of HEK293...transient expression of a gene of interest in cell culture. This protocol describes how to transfect suspension... cells are considered biosafety level 2. Please ensure that you are in compliance with your institution...the DNA and working stock of PEI-MAX to room temperature before use, and warm the BalanCD HEK293 transfection...water to a final volume of 1 L and recheck pH to ensure that it has not drifted. Filter the solution through...biosafety cabinet and incubate for 1 h at room temperature. After the BCD TFX has warmed in the bead bath...

  4. Centrifugation

    Type
    Protocol
    ...samples at specific temperatures. This protocol will cover the general procedure and features to keep in mind...densities is essential to many different experimental procedures, such as minipreps and RNA extractions. Plus,... centrifuge may have specific instructions so be sure to check in with someone familiar with your lab’...the time and speed needed for your experiment. Procedure An example of a balanced tabletop centrifuge, ...a tabletop microcentrifuge like the one in the picture above. Wear PPE appropriate for the lab space in.... Before using the centrifuge for your samples, ensure that the centrifuge is clean and that everything...center (see image to right). Even distribution ensures that the centrifuge is balanced. Using the centrifuge...

  5. Protocols for Molecular Biology, Plasmid Cloning, and Viral Preps

    Type
    Protocol
    ...the building blocks for many more complicated procedures. Plasmid Cloning Protocols for constructing and...Biosafety Levels One and Two (BSL-1 and BSL-2) Safety measures for laboratories operating at BSL-1 and BSL-2 ...to Video Making LB Agar Plates Create plates to culture bacteria in the lab Watch the Video! Over-Agar ...Antibiotic Plating Quickly add antibiotic to a pre-poured plate Watch the Video! Streaking Bacteria Isolate...Watch the Video! Inoculating a Liquid Bacterial Culture Prepare and grow bacteria in liquid medium Watch...extract, and precipitate DNA DNA Quantification Measure DNA concentration with a spectrophotometer Restriction...Video! Sequence Analysis Verify important plasmid features using sequence analysis RNA Extraction Without...

  6. DNA Quantification

    Type
    Protocol
    ...and click measure, be sure to record the concentration and purity. Note: Purity is measured under the ...Inoculating a Liquid Bacterial Culture Recovering Plasmid DNA from Bacterial Culture Restriction Digest of Plasmid...substance in that liquid. In order to accurately measure the concentration of a substance based on its absorbance... of whether you have a NanoDrop, follow the manufacturer's instructions for the spectrophotometer specific...Spectrophotometer Tips Before measuring any samples, be sure to ‘blank’ the spectrophotometer using the solution...resuspended in, but with no DNA added. 'Blanking' measures the background inherent to the machine and your...your solvent. If using a NanoDrop to measure your samples, place 1-2µL of mini-prepped DNA onto the pedestal...

  7. Kit Free RNA Extraction

    Type
    Protocol
    ...list above. Procedure Option #1 - Solution D Protocol Before starting this protocol, make sure to prepare...TRIzol® User Guide from ThermoFisher Scientific . Figure 1: A diagram of the different steps in RNA extraction...step, Option B) Glycogen (Optional) Caution Make sure to read the SDS (Safety Data Sheet) for safety warnings...use 1 mL of Solution D per 100 mg of cells. For cultured cells: use 1 mL of Solution D per 1 X 10 7 cells...cells. Allow sample(s) to sit at room temperature for 5 minutes to allow for dissociation of the nucleoprotein...centrifuge, bring this equipment back to room temperature, as prolonged storage in the cold room may damage...top, aqueous phase to a new RNAse-free tube. The mixture separates into a bottom organic layer, an interphase...

  8. Protocol - How to Streak a Plate

    Type
    Protocol
    ...DNA from Bacterial Culture Introduction If you have a glycerol stock or stab culture of bacteria and need...inoculate a bacterial culture for DNA purification will minimize the chance of having a mixture of plasmids in...touch the bacteria growing within the punctured area of the stab culture or the top of the glycerol stock.... (with appropriate antibiotic) Bacterial stab Procedure Obtain an LB agar plate with appropriate antibiotic...sterilize it by passing it through a flame, just be sure to allow enough time for the loop to cool before...diagonal lines in another section of the plate. Make sure that the first line (and only the first) in each...plasmids, which sometimes recombine at 37 °C. Be sure to check this before incubating your plate. In the...

  9. Weighing Reagents Protocol

    Type
    Protocol
    ...reagents for a stock mixture is an essential laboratory technique, as imprecise measurements can affect the ...other solutions. A key part of this task is making sure you’re weighing all reagents precisely to create....) Reagents Material that will be weighed out Procedure The balance in the photo above has a capacity ...need to weigh it out in the fume hood to prevent exposure. Read the reagent’s material safety data sheet...by looking for a weight range on the scale. Make sure that the weight of the material you’re weighing ...weight of the weighing boat or paper into your measurement. Hit the tare button (or zero button) to set ... material directly to the tube to weigh it. Make sure that your scoopula or spatula is clean, and sterile...

  10. Protocol - How to Run an Agarose Gel

    Type
    Protocol
    ...New England Biolabs B7022S Procedure Pouring a Standard 1% Agarose Gel: Measure 1 g of agarose. Pro-Tip ...pipette tip. Place newly poured gel at 4 °C for 10-15 mins OR let sit at room temperature for 20-30 mins, until...Introduction Gel electrophoresis is the standard lab procedure for separating DNA by size (e.g., length in base...very little difference between the two. Note: Make sure to use the same buffer as the one in the gel box...earlier so that it is already cold when the gel is poured into it. Loading Samples and Running an Agarose...loading the sample in the well, maintain positive pressure on the sample to prevent bubbles or buffer from...DNA ladder in the first lane as a guide (the manufacturer's instruction will tell you the size of each ...

  11. Plasmid Cloning by PCR (with Protocols)

    Type
    Protocol
    ...but you should ensure that the bases do not result in the formation of a hairpin structure within your primer...You should select an annealing temperature based on the melting temperature (Tm) of the portion of the primer...TGCTTAGCGGCCGCTCAGTACTTCGAGATATGCCA-3’. Experimental Procedure Run PCR and Ppurify the PCR Product Run PCR to...you can use a pretty wide range of annealing temperatures, but you may need to increase your primer length... phosphatase) are commonly used. Follow the manufacturer’s instructions. Isolate Your Insert and Vector...Proceed with the transformation according to the manufacturer’s instructions for your competent cells. For ...colonies, you should conduct a positive control to ensure that your transformation worked. You could also...

  12. Protocol - Bacterial Transformation

    Type
    Protocol
    ...with their competent cells) to ensure that your transformation procedure is working. TIP: Sometimes less... competent cells. This is a relatively simple procedure and is useful for performing low efficiency transformations...media Competent cells DNA you'd like to transform Procedure Take competent cells out of -80°C and thaw on ... from storage at 4°C and let warm up to room temperature and then (optional) incubate in 37°C incubator...control plasmid. Incubate the competent cell/DNA mixture on ice for 20-30 mins. Heat shock each transformation...allowing you to recover all transformants. If the culture volume is too big, gently collect the cells by ...electro-magnetic field is applied to the cell/DNA mixture to induce membrane permeability. To do this you...

  13. Gibson Assembly Protocol

    Type
    Protocol
    ...DNA fragments at once. Procedure Design your plasmid and order primers (see figure to the right). When designing...sequences on the ends (sequences A and B in the figures). These identical sequences can be created via ...to the target sequence. Avoid strong secondary structures in the homology region. Hairpins in this region...three different enzymes within a single buffer mixture and an optional SSB protein to improve accuracy...Exonuclease and furthermore reduces secondary structure of ssDNA. (Rabe & Cepko, 2020). Incubate the mix... mix for 1 hour at 50 °C or follow manufacturer's instructions. You can purchase master mix or make your... DNA molecules up to several hundred kilobases. Nature Methods , 6(5), 343–345. https://doi.org/10.1038...

  14. Plasmid Cloning by Restriction Enzyme Digest (with Protocols)

    Type
    Protocol
    ...buffer, it will save you time in future steps. Experimental Procedure Digest your DNA Set up restriction...subcloning), including design and experimental procedures. Protocols...orientation on your target vector. If you are not sure what vector to use, you can check out our Empty ... for short). You might need to express YGOI in cultured mammalian cells. The problem is that the only ... phosphatase) are commonly used. Follow the manufacturer’s instructions. Isolate your insert and vector...your bacterial strain of choice. Follow the manufacturer’s instructions for your competent cells. For ...colonies, you should conduct a positive control to ensure that your transformation worked. You should also...

  15. Antibody Validation Using the Indirect ELISA Method

    Type
    Protocol
    ...Foil Before Starting Warm reagents to room temperature. Procedure Section 1: Prepare the Antigen Standard...incubating certain steps at 4 °C instead of room temperature or 37 °C. The protocol notes when there are different...microplate shaker set to 400 rpm for 2 h at room temperature or overnight at 4 °C . Section 3: Primary antibody...microplate shaker set to 400 rpm for 2 h at room temperature or overnight at 4 °C . Section 4: Secondary antibody...microplate shaker set to 400 rpm for 2 h at room temperature or overnight at 4 °C . Section 5: TMB reaction...seal and wrap in foil. Incubate plate at room temperature on a microplate shaker set at 400 rpm for 15–...Solution directly to the wells to stop the reaction. Measure the absorbance of each well at 450 nm on the spectrophotometer...

  16. Immunocytochemistry

    Type
    Protocol
    ...the basic steps for fixing and staining cells in culture with a primary antibody against a target protein...the basic steps for fixing and labeling cells in culture with a primary antibody against a target protein... conical tubes Before Starting Refer to the manufacturer's instructions for additional information specific...stock solution into 5 mL PBS. Protect from light. Procedure Section 1: Seeding cells Place a sterile poly-...coated coverslip in each well of a 24-well cell culture treated plate. Seed 5 x 10 3 HeLa cells per well...platform. Permeabilize cells for 10 min at room temperature ( RT ) on a rocking platform in 500 µL permeabilization...concentration will vary between antibodies. Review the manufacturer's instructions before starting your experiment...

  17. AAV Titration by qPCR Using SYBR Green Technology

    Type
    Protocol
    ...between them. Sample Data Figure 1: Example of a valid 8-point standard curve. Figure 2: Example of the amplification...additional 10 samples (n+10 – the additional amount will ensure that there is enough master mix for all samples...0.15 μL 15 μL Nuclease Free Water 4.7 μL 470 μL Procedure Prepare a plasmid stock of 2 x 10 9 molecules/... of the sample dilution series is critical. Make sure to pipet each dilution up and down at least 10 times...standard curve and the sample dilutions. Pro-Tip Make sure that the qPCR is valid by checking to the following... be seen. The presence of a second peak at a temperature of ~70–75 °C usually indicates the presence of...

  18. AAV Production in HEK293 Cells

    Type
    Protocol
    ...solution to mix for 10 min and then recheck the pH to ensure that it has not drifted. Filter the solution through.... Thaw a new vial of cells after 30 passages. Procedure Trypsinize and resuspend the HEK293T cells from...80% confluence. For each T-175 flask: Aspirate culture media and rinse once with 10 mL of PBS. Aspirate.... Cells should be at ~80% confluence. Aspirate culture media and rinse once with 60 mL PBS. Aspirate PBS...the OptiMEM + DNA + PEI solution to the CS5. Make sure that all five layers are covered with media. 293T...should always be added away from the cells (not poured on them) and can be adjusted by carefully tilting...proceeding with the purification protocol. Sample Data Figure 1: HEK293T cells at various confluencies. This ...

  19. Protocol - How to Ligate Plasmid DNA

    Type
    Protocol
    ...Incubate at room temperature for 2hr, or at 16°C overnight (following the manufacturer’s instructions). ...end and a different enzyme on the 3' end). This ensures that the insert will be added in the correct orientation...reaction, scale the reaction size as necessary - being sure to increase the amount of buffer proportionally....C. Whenever you need to set up ligations in the future you can thaw a new tube that you know has only ...using "high concentration" ligase, 5min at room temperature is enough. For trickier ligations (such as ligation...

  20. Protocol - How to Purify DNA from an Agarose Gel

    Type
    Protocol
    ... the UV exposure of the DNA. Therefore, it is a bad idea to use a gel imager to take a picture of the ...isolate and purify DNA fragments based on size. The procedure starts with standard agarose gel electrophoresis...have run your gel, move it to an open UV box (be sure to wear proper UV protection - especially for your...QIAquick Gel Extraction Kit . Always follow the manufacturer's instructions. Note: It is usually important...
Showing: 21 - 40 of 50 results