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Revision as of 12:41, 23 August 2019

Sci-Phi 29

  • General
    • Antibiotic Stock Solution
      1. Weigh X gram of the antibiotic of interest.
      2. Dissolve the antibiotic of interest in milli-Q.

      Note: Dissolve in 95% ethanol in case of Chloramphenicol.

    • Colony PCR

      Children will automatically close upon closing its parent.

    • DNA Gel Electrophoresis

      Protocol for DNA Gel Electrophoresis includes mutagenic chemical such as EtBR, SYBR Safe or another DNA staining. Carry the protocol out in the assigned area and wear gloves.



      Gel preparation:
      1. Prepare 0.8% (w/v) agarose in TAE/TBE buffer.
      2. Heat the gel solution to dissolve the agarose in the microwave. Swirl the solution a few times while heating to ensure that the agarose powder is dissolved. The agarose is dissolved when the solution has bubbles and is clear.

      Casting of gel:
      1. Pick an appropriate gel stand and gel comb and place in the gel casting stand. Make sure to fasten the stand in place. Perform the next few steps quickly to prevent the gel from solidifying.
      2. Spread 15 µl (large stand)/ 4-5 µl (small stand) of SYBRSafe/EtBr on the stand.
      3. Pour the gel carefully into the stand. Ensure that 3/4th of the teeth of the comb is submerged in the solution.
      4. Allow the gel to solidify for about 30 minutes.

      Sample loading:
      1. After the gel solidifies, remove the comb carefully and transfer the gel to the gel box filled with TAE/TBE buffer.
      2. Cut a strip of parafilm. Spot loading dye on the parafilm (1:5::dye:sample) and add the sample. (E.g. 5µl of sample and 1µl of dye.) Use the pipette to mix the solutions.
      3. Load the samples carefully in the slots. Ensure not to poke the gel.
      4. Load 3 µl of DNA ladder.
      5. Set the voltage to 100 Volt.
      6. Run the gel till the DNA has ran 2/3 of the gel which equals approximately 45 minutes.
      7. Visualize the gel using GelDsoc.
    • DNA Gel Purification

      Children will automatically close upon closing its parent.

    • Gibson Assembly

      Children will automatically close upon closing its parent.

    • Ligation

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    • Nanodrop DNA quantification
      1. Use a nanodrop spectrometer to quantify the DNA amount and purity in a DNA purification or extraction sample.
      2. Adjust the settings to measure dsDNA at 260nm and impurities at 230 nm and 280 nm.

      Note: DNA should have a 260/280 between 1.8-2.0. The sample is contaminated if the value is below 1.8.

    • PCR Clean-Up

      Children will automatically close upon closing its parent.

    • Plasmid Isolation

      Protocol for plasmid isolation using Promega PureYield MiniPrep Kit:

      Prepare lysate:
      1. Pipette 1.5ml of the bacterial culture in an eppendorf tube and centrifuge culture for 30 seconds at maximum speed in a microcentrifuge.
      2. Discard the supernatant.
      3. Repeat Steps 1 and 2 until the entire inoculum volume has been spun down.
      4. Add 600µl of MilliQ water to the cell pellet, and resuspend completely.
      5. Add 100µl of Cell Lysis Buffer, and mix by inverting the tube 6 times. The solution should change from opaque to clear blue, indicating complete lysis.
        6. Note: if you have more sample volume, split it. This will help complete this step quickly.
        Note: Proceed to Step 6 within 2 minutes. Excessive lysis can result in denatured plasmid DNA. If processing a large number of samples, process samples in groups of ten or less. Continue with the next set of ten samples after the first set has been neutralized and mixed thoroughly.
      6. Add 350µl of cold (4–8°C) Neutralization Solution, and mix thoroughly by inverting the tube. The sample will turn yellow when neutralization is complete, and a yellow precipitate will form. Invert the sample an additional 3 times to ensure complete neutralization.
      7. Centrifuge at maximum speed in a microcentrifuge for 3 minutes.
      8. Transfer the supernatant (~900µl) to a PureYield™ Minicolumn. Do not disturb the cell debris pellet. For maximum yield, transfer the supernatant with a pipette.
      9. Place the minicolumn into a PureYield™ Collection Tube, and centrifuge at maximum speed in a microcentrifuge for 15 seconds or 30 seconds.
      10. Discard the flow through, and place the minicolumn into the same PureYield™ Collection Tube.

      Wash:
      1. Add 200µl of Endotoxin Removal Wash to the minicolumn. Centrifuge at maximum speed in a microcentrifuge for 15 seconds. It is not necessary to empty the PureYield™ Collection Tube.
      2. Add 400µl of Column Wash Solution to the minicolumn. Centrifuge at maximum speed in a microcentrifuge for 30 seconds.

      Elute:
      1. Transfer the minicolumn to a clean 1.5ml microcentrifuge tube, then add 30µl of nuclease free water at neutral pH directly to the minicolumn matrix. Let stand for 1 minute at room temperature.
        2. Notes: 1. Elution buffer can also be used, but it can interfere with future cloning steps. 2. For large plasmids (>10kb), warm the Elution Buffer to 50ºC prior to elution, and increase elution volume to 50µl. Also incubate the column at room temperature (22–25°C) for 5–10 minutes before proceeding to Step 14.
      2. Centrifuge at maximum speed in a microcentrifuge for 15 seconds to elute the plasmid DNA. Cap the microcentrifuge tube, and store eluted plasmid DNA at –20°C.
      3. Follow the nanodrop quantification protocol to determine the purity and yield of your plasmid isolation.
    • Restriction Digestion

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  • Strain Specific
    • Bacillus Subtillis
      • Electrocompetent Cells Preparation

        As long as the inner element has inner as one of its classes then it will be toggled.

        Lorem ipsum dolor sit amet, consectetur adipiscing elit. Maecenas tempus placerat fringilla. Duis a elit et dolor laoreet volutpat. Aliquam ultrices mauris id mattis imperdiet. Aenean cursus ultrices justo et varius. Suspendisse aliquam orci id dui dapibus blandit. In hac habitasse platea dictumst. Sed risus velit, pellentesque eu enim ac, ultricies pretium felis.

      • Glycerol Stock

        As long as the inner element has inner as one of its classes then it will be toggled.

        Lorem ipsum dolor sit amet, consectetur adipiscing elit. Maecenas tempus placerat fringilla. Duis a elit et dolor laoreet volutpat. Aliquam ultrices mauris id mattis imperdiet. Aenean cursus ultrices justo et varius. Suspendisse aliquam orci id dui dapibus blandit. In hac habitasse platea dictumst. Sed risus velit, pellentesque eu enim ac, ultricies pretium felis.

      • LB Agar preparation

        As long as the inner element has inner as one of its classes then it will be toggled.

        Lorem ipsum dolor sit amet, consectetur adipiscing elit. Maecenas tempus placerat fringilla. Duis a elit et dolor laoreet volutpat. Aliquam ultrices mauris id mattis imperdiet. Aenean cursus ultrices justo et varius. Suspendisse aliquam orci id dui dapibus blandit. In hac habitasse platea dictumst. Sed risus velit, pellentesque eu enim ac, ultricies pretium felis.

      • Liquid Broth (LB) Medium Preparation

        As long as the inner element has inner as one of its classes then it will be toggled.

        Lorem ipsum dolor sit amet, consectetur adipiscing elit. Maecenas tempus placerat fringilla. Duis a elit et dolor laoreet volutpat. Aliquam ultrices mauris id mattis imperdiet. Aenean cursus ultrices justo et varius. Suspendisse aliquam orci id dui dapibus blandit. In hac habitasse platea dictumst. Sed risus velit, pellentesque eu enim ac, ultricies pretium felis.

      • Electroporation

        As long as the inner element has inner as one of its classes then it will be toggled.

        Lorem ipsum dolor sit amet, consectetur adipiscing elit. Maecenas tempus placerat fringilla. Duis a elit et dolor laoreet volutpat. Aliquam ultrices mauris id mattis imperdiet. Aenean cursus ultrices justo et varius. Suspendisse aliquam orci id dui dapibus blandit. In hac habitasse platea dictumst. Sed risus velit, pellentesque eu enim ac, ultricies pretium felis.

    • Escherichia coli
      • Chemically Competent Cells Preparation

        The protocol for chemical competent cells has a duration of three days, starting from the preparation of the reagents. Throughout the protocol, it is mandatory to work under aseptic conditions to prevent contamination risks as much as possible.

          Day 1 (preparation):
        1. Prepare the necessary reagents and sterilize the reagents and centrifuge bottles.
          • 1 L Luria Broth (LB) medium
          • 10 mL Luria Broth (LB) medium
          • 400 mL 100mM CaCl2
          • 20 mL 100 mM CaCl2+ 15% glycerol
        2. Store the CaCl2 and the CaCl2 + 15% glycerol stock solution at 4°C for at least 24 h.
        3. Take the strain of interest from the -80°C and store it on ice.
        4. Streak out the desired E. coli strain on LB plate with antibiotic selection marker and incubate at 37°C overnight.

          5. Day 2 (preparation):
        5. Inoculate one single colony from the overnight grown plate in 10 mL LB medium with antibiotic selection marker overnight at 37°C while shaking at 200 rpm.

          6. Day 3:
        6. Inoculate 10 mL from the overnight grown liquid cell culture in 1 L of LB medium with antibiotic selection marker and grow the inoculated medium at 37°C while shaking at 200 rpm until the OD600 is between 0.4 and 0.6.The OD is measured the first time after 1.5 hours after inoculation. (Important: the OD should not rise above 0.6 in order to obtain good competent cells)

          7. Important notes:
          • From this point be very careful with your cells. The cells are very fragile.
          • Everything that touches the cells should not be warmer than 4°C.
          • Keep the cells always on ice. Thus also resuspension steps needs to be performed on ice.
          • Work clean and fast, the cells heat up very quickly which influence the quality of the competent cells a lot.
        7. Split the 1 L LB medium with cells into the centrifuge cylinders by weighing them in order to have equal weight distribution during centrifuging. If necessary prepare a counterweight for in the centrifuge.
        8. Harvest the cells by centrifugation for 5 minutes at 4000 rpm at 4°C and resuspent all of the pellets carefully in 200 mL ice cold CaCl2. Use an electronic pipet to resuspend the pellet.
        9. Incubate the resuspended cells for 20 minutes on ice.
        10. Harvest the resuspended cells by centrifugation for 5 minutes at 3000 rpm at 4°C, and resuspent the new pellet gently in 100 mL ice cold CaCl2. Use an electronic pipet to resuspend the pellet.
        11. Incubate the resuspended cells for 1 hour on ice. In the meantime mark approximately 75 1.5 mL Eppendorf tubes and store them on ice.
        12. Harvest the resuspended cells by centrifugation for 5 minutes at 3000 rpm at 4°C, and resuspent the new pellet gently in 10 mL ice cold CaCl2 + 15% glycerol. Use an electronic pipet to resuspend the pellet.
        13. Aliquot 50 μL into sterile 1.5 mL microfuge tubes and snap freeze with liquid nitrogen. (Since the cells are very fragile, cut the pipettips in order to reduce the stress on the cells.)
        14. Store frozen cells in the -80°C freezer.
        15. Immediately perform a heat shock transformation to determine the transformation efficiency.
      • Glycerol Stock
        1. Incubate ~50 µl cells in 5 ml LB and selection marker overnight.
        2. Pipette 1.5 ml in an eppendorf tube and spin down the bacterial culture.
        3. Discard the supernatant.
        4. Repeat step 2 and 3
        5. Resuspend the pellet in 0.9 ml liquid bacterial culture.
        6. Add the liquid bacterial culture from eppendorf tube to cryotube.
        7. Add 0.9 ml 50% glycerol to the cryotube and mix well.
        8. Store at -80°C.
      • LB Agar preparation
        1. Dissolve 14.24g LB agar in 400 mL of milli-Q.
        2. Autoclave the LB agar solution.
      • Liquid Broth (LB) Medium Preparation
        1. Dissolve 20 g/L Luria Broth (LB) powder in water as described in the instructions provided by the manufacturer.
        2. Autoclave the medium at 121°C to heat sterilize.
        3. Store the sterilised LB medium at room temperature.
      • Heat Shock Transformation
        1. Thaw 50 µL of chemical competent cells on ice for 10 minutes.
        2. Add 0.1- 10 ng plasmid DNA or 20 - 50 ng ligate to the competent cells. incubate on ice for 30 minutes.
        3. Heat shock 45 seconds at 42°C.
        4. Incubate 2 minutes on ice.
        5. Add 1 mL LB medium, mix gently.
        6. Incubate 1 hour at 37°C.
        7. Prepare agar plate with the right corresponding antibiotic marker. 25 mL agar with 200 µL of antibiotic stock solution.
        8. Plate 100 µL of cell suspension on the agar plate and grow it overnight at 37°C.
    • Pseudomonas Putida
      • Electrocompetent Cells Preparation

        As long as the inner element has inner as one of its classes then it will be toggled.

        Lorem ipsum dolor sit amet, consectetur adipiscing elit. Maecenas tempus placerat fringilla. Duis a elit et dolor laoreet volutpat. Aliquam ultrices mauris id mattis imperdiet. Aenean cursus ultrices justo et varius. Suspendisse aliquam orci id dui dapibus blandit. In hac habitasse platea dictumst. Sed risus velit, pellentesque eu enim ac, ultricies pretium felis.

      • Glycerol Stock

        As long as the inner element has inner as one of its classes then it will be toggled.

        Lorem ipsum dolor sit amet, consectetur adipiscing elit. Maecenas tempus placerat fringilla. Duis a elit et dolor laoreet volutpat. Aliquam ultrices mauris id mattis imperdiet. Aenean cursus ultrices justo et varius. Suspendisse aliquam orci id dui dapibus blandit. In hac habitasse platea dictumst. Sed risus velit, pellentesque eu enim ac, ultricies pretium felis.

      • LB Agar preparation

        As long as the inner element has inner as one of its classes then it will be toggled.

      • Liquid Broth (LB) Medium Preparation

        As long as the inner element has inner as one of its classes then it will be toggled.

        Lorem ipsum dolor sit amet, consectetur adipiscing elit. Maecenas tempus placerat fringilla. Duis a elit et dolor laoreet volutpat. Aliquam ultrices mauris id mattis imperdiet. Aenean cursus ultrices justo et varius. Suspendisse aliquam orci id dui dapibus blandit. In hac habitasse platea dictumst. Sed risus velit, pellentesque eu enim ac, ultricies pretium felis.

      • Electroporation

        As long as the inner element has inner as one of its classes then it will be toggled.

        Lorem ipsum dolor sit amet, consectetur adipiscing elit. Maecenas tempus placerat fringilla. Duis a elit et dolor laoreet volutpat. Aliquam ultrices mauris id mattis imperdiet. Aenean cursus ultrices justo et varius. Suspendisse aliquam orci id dui dapibus blandit. In hac habitasse platea dictumst. Sed risus velit, pellentesque eu enim ac, ultricies pretium felis.

    • Vibrio Natriegens
      • Electrocompetent Cells Preparation

        As long as the inner element has inner as one of its classes then it will be toggled.

        Lorem ipsum dolor sit amet, consectetur adipiscing elit. Maecenas tempus placerat fringilla. Duis a elit et dolor laoreet volutpat. Aliquam ultrices mauris id mattis imperdiet. Aenean cursus ultrices justo et varius. Suspendisse aliquam orci id dui dapibus blandit. In hac habitasse platea dictumst. Sed risus velit, pellentesque eu enim ac, ultricies pretium felis.

      • Glycerol Stock

        As long as the inner element has inner as one of its classes then it will be toggled.

        Lorem ipsum dolor sit amet, consectetur adipiscing elit. Maecenas tempus placerat fringilla. Duis a elit et dolor laoreet volutpat. Aliquam ultrices mauris id mattis imperdiet. Aenean cursus ultrices justo et varius. Suspendisse aliquam orci id dui dapibus blandit. In hac habitasse platea dictumst. Sed risus velit, pellentesque eu enim ac, ultricies pretium felis.

      • LB Agar preparation

        As long as the inner element has inner as one of its classes then it will be toggled.

        Lorem ipsum dolor sit amet, consectetur adipiscing elit. Maecenas tempus placerat fringilla. Duis a elit et dolor laoreet volutpat. Aliquam ultrices mauris id mattis imperdiet. Aenean cursus ultrices justo et varius. Suspendisse aliquam orci id dui dapibus blandit. In hac habitasse platea dictumst. Sed risus velit, pellentesque eu enim ac, ultricies pretium felis.

      • Liquid Broth (LB) Medium Preparation

        As long as the inner element has inner as one of its classes then it will be toggled.

        Lorem ipsum dolor sit amet, consectetur adipiscing elit. Maecenas tempus placerat fringilla. Duis a elit et dolor laoreet volutpat. Aliquam ultrices mauris id mattis imperdiet. Aenean cursus ultrices justo et varius. Suspendisse aliquam orci id dui dapibus blandit. In hac habitasse platea dictumst. Sed risus velit, pellentesque eu enim ac, ultricies pretium felis.

      • Electroporation

        As long as the inner element has inner as one of its classes then it will be toggled.

        Lorem ipsum dolor sit amet, consectetur adipiscing elit. Maecenas tempus placerat fringilla. Duis a elit et dolor laoreet volutpat. Aliquam ultrices mauris id mattis imperdiet. Aenean cursus ultrices justo et varius. Suspendisse aliquam orci id dui dapibus blandit. In hac habitasse platea dictumst. Sed risus velit, pellentesque eu enim ac, ultricies pretium felis.