Team:HKU-Hong Kong/Lab Protocol

From 2011.igem.org

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Colony PCR
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<LI>Add the following reagents into a PCR tube (in order) and mix well.</LI>
<LI>Add the following reagents into a PCR tube (in order) and mix well.</LI>

Revision as of 11:08, 4 October 2011

Lab Protocol
A. DNA WORK
Agarose Gel Electrophoresis
  1. Preparation of agarose gel
    1. Pour 100 mL of 1X TAE buffer into a conical flask.
    2. Add the agarose powder to the buffer in the amount with respect to the concentration of the agarose solution (e.g. add 1 g for preparing 1% agarose gel solution).
    3. Use a plastic wrap to cover the opening of the conical flask and microwave for approximately 2 minutes or until the agarose dissolves completely.
    4. Pour the agarose solution into another conical flask which specifies for holding ethidium bromide (EB) – containing solution.
    5. Add 1 – 2 ul of EB into the agarose solution and mix well.
    6. Pour the solution into a gel tray with a comb. Remove any bubbles formed.
    7. Allow the gel to solidify which takes approximately 30 minutes.
    8. Discard all the wastes into the EB waste box.
  2. Electrophoresis
    1. Remove the comb and place the solidified gel into the electrophoresis tank.
    2. Add TAE buffer to the tank when necessary.
    3. Add 6X loading buffer to the DNA sample in the ratio of 1:6 and mix well.
    4. Load the samples into the wells with care.
    5. Load 2-3 ul of marker to a well for reference.
    6. Run the electrophoresis at around 140V for about 30 minutes.
    7. Take the gel photo in the UV-illuminating machine.
DNA Extraction from Agarose Gel
  1. Gel extraction
    1. Wear UV protection glasses before the gel extraction.
    2. Place the gel onto the transilluminator.
    3. Turn on the transilluminator and quickly cut the desired gel band.
    4. Place the cut band into an eppendorf tube for further processing.
    5. Discard all the wastes into the EB waste box.
  2. DNA extraction (Adopt from Qiagen)
    1. Weight the Eppendorf tube and determine the weight of the cut band.
    2. Add 3 volumes of extraction buffer to 1 volume of the cut get.
    3. Place the Eppendorf tube (with the cut gel and the extraction buffer) into 55oC water bath to dissolve all the agarose gel.
    4. After dissolving, add the mixture to the spin column with a collection tube.
    5. Centrifuge at 11,000 rpm for 1 minute.
    6. Discard flow through.
    7. Add 750 ul of washing buffer and centrifuge at 11,000 rpm for 1 minute.
    8. Discard the flow through and centrifuge again at 11,000 rpm for 1 minute.
    9. Place the collection tube to a new Eppendorf tube.
    10. Add 20 mL elution buffer directly to the centre of the membrane of the collection tube.
    11. Let it stand for approximately 3 minutes.
    12. Centrifuge at 11,000 rpm for 1 minutes and collect the flow through (i.e. product).
    13. Take a small portion of the DNA product for confirmation by gel electrophoresis.
  • Protocol adopt from http://www.qiagen.com/literature/render.aspx?id=201083
    • DNA Digestion
    1. Add the following reagents, with the enzymes added at the last, into a tube.
    2. All steps should be carried out on ice.
    3. Mix well after addition of all the reagent.
    4. Incubate the mixture at 37oC for several hours.
    Miniprep(Adopt from Qiagen)
    1. Centrifuge the sample at 8,000 rpm for 1 minute.
    2. Discard the supernatant.
    3. Add 250 ul P1 buffer to resuspend the pellet (tap to suspend the pellet completely).
    4. Add 250 ul P2 buffer and mix gently by inverting the tube for several times.
    5. Add 350 ul N3 buffer and mix thoroughly. The solution should now turn cloudy.
    6. Centrifuge the solution at 13,000 rpm for 10 minutes.
    7. Transfer the supernatant to a spin column with a collection tube inside.
    8. Centrifuge at 12,500 rpm for 1 minute. Discard the flow through.
    9. Add 750 ul PE buffer to the collection tube and centrifuge at 12,500 rpm for 1 minute.
    10. Discard the flow through and centrifuge again to remove all remaining washing buffer.
    11. Place the collection tube into a new eppendorf tube.
    12. Add 50 ul elution buffer directly at the centre of the membrane of the collection tube.
    13. Let it stand for approximately 3 minutes.
    14. Centrifuge at 12,500 rpm for 1 minutes and collect the flow through (i.e. the product).
  • Protocol adopt from: http://www.qiagen.com/literature/render.aspx?id=201081
    • Polymerase Chain Reaction
      Colony PCR
      1. Add the following reagents into a PCR tube (in order) and mix well.
      2. Set the following PCR program.
    1. Reverse PCR
      1. Add the following reagents into a PCR tube (in order) and mix well.
      2. Set the following PCR program.
    2. Overlap PCR
      1. First, two PCR reactions are set for amplifying the two genes, tetR and HNS, separately.
      2. Add the following reagents into a PCR tube (in order) and mix well.
      3. Set the following PCR program.
      4. Set up another PCR reaction using a primer with a linker to link the two genes.
      5. Add the following reagents into a PCR tube (in order) and mix well.
      6. Set the following PCR program.
      7. Set up another PCR reaction to further amplify the fused product.
      8. Add the following reagents into a PCR tube (in order) and mix well.
      9. Set the following PCR program.
    DNA ligation
    1. Add the following reagents, with the enzymes added at the last, into a tube.
    2. Incubate at 16oC overnight.
    Sequencing
    1. Send to BGI company for sequencing.
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