Team:Guanajuato Mx/Notebook/Protocols

iGEM Guanajuato Mx

MOLECULAR LAB
PATCH LAB
PROTOCOLS

NOTEBOOK

PROTOCOLS

Fast-n-Easy Plasmid Mini-Prep Kit

Column based isolation of plasmid DNA
Protocol of Jena Bioscience

  1. Cell Harvest and Lysis
    • Harvesting of the bacterial cell culture (1-3 ml) by centrifugation.
    • Resuspend pelleted bacterial cells in 300 Lysis Buffer by pipetting or vortexing for 1 min.
  2. Neutralization
    • Add 300 of Neutralization Buffer (containing RNase A) to sample and mix gently by inverting the tube 4-6 times (do not vortex).
    • Centrifuge at 10,000 g for 5 min at room temperature in a microcentrifuge.
    • The color of the binding mixture should change to bright yellow indicating a pH of 7.5 required for optimal DNA binding. An orange or violet color shows a pH >7.5 and indicates an inefficient DNA adsorption. In this case, it is recommended to adjust the pH of the mixture by addition of a small volume of 3 M sodium acetate, pH 5.0 before proceeding.
  3. Column Activation
    • Place a Binding Column into a 2 mL collection tube.
    • Add 100 μl of Activation Buffer into the Binding Column.
    • Centrifuge at 10,000 g for 30 sec in a microcentrifuge.
  4. Column Loading
    • Apply the supernatant from step 2 into the activated Binding column by decanting or pipetting.
    • Centrifuge at 10,000 g for 30 sec.
    • Discard the flow-through.
  5. Column Washing
    • Place the DNA loaded Binding Column into the used 2 mL tube.
    • Apply 500 μl of the Washing Buffer to the Binding Column.
    • Centrifuge at 10,000 g for 30 sec and discard the flow-through.
    • Optional Secondary Washing: Recommended only for DNA >200 bp, if highly purified DNA (for DNA sequencing,transfection etc.) is required.
      • Add 700 μl of Washing Buffer to the Binding Column.
      • Centrifuge at 10,000 g for 30 sec and discard the flow-through.
      • Centrifuge again for 2 min to remove residual Washing Buffer.
  6. Elution
    • Place the Binding Column into a clean 1.5 mL microtube.
    • Add 30-50 μl Elution Buffer or add-water to the center of the column membrane.
    • Centrifuge at 10,000 g for 1 min to elute DNA.

Dialysis of the ligation product

  1. Place approximately 10 mL of deionized water or distilled water in a petri dish
  2. Place a millipore filter on top of the water.
  3. Place the ligation product onto the center of the millipore filter
  4. Let it dialyse for 15 minutes
  5. Carefully retrieve the DNA with a micropipette and place it in a microcentrifuge tube

PCR Purification Kit

Spin-column based DNA cleanup from PCr samples
Protocol of Jena Bioscience

  1. Standard Samples Preparation
    • For DNA fragment sizes in the range of 200 bp to 5 kbp, add 5 volumes of Binding Buffer to 1 volume of DNA sample and mix well.
    • For DNA fragment sizes smaller than 200 bp or larger than 5 kbp, add 3 volumes Binding Buffer and 2 volumes of isopropanol to the PCR sample.
  2. Column Activation
    • Place a Spin Column into a 2ml collection tube.
    • Add 100 μl of Activation Buffer into the Spin Column.
    • Centrifuge at 10,000 g for 30 sec in a microcentrifuge.
  3. Column Loading
    • Apply the sample mixture from step 1 into the activated Spin Column.
    • Centrifuge at 10,000 g for 30 sec in a microcentrifuge.
    • Discard the flow-through.
  4. Column Washing
    • Place the DNA loaded Spin Column into the used 2 ml tube.
    • Apply 700 μl of Washing Buffer to Spin Column.
    • Centrifuge at 10,000 g for 30 sec and discard the flow-through.
    • Optional Secondary Washing: Recommended only for DNA 200 bp, if highly purified DNA (for DNA sequencing,transfection etc.) is required.
      • Add 700 μl of Washing Buffer to the Spin Column.
      • Centrifuge at 10,000 g for 30 sec and discard the flow-through.
      • Centrifuge again for 2 min to remove residual Washing Buffer.
  5. Elution
    • Place the Spin Column into a clean 1.5 ml microtube.
    • Add 30-50 μl Elution Buffer or add water to the center of the column membrane.
    • Incubate at room temperature for 1 min.
    • Centrifuge at 10,000 g for 1 min to elute DNA.

Extra procedure for human Lysozyme extraction in from blood leukocytes: Modified from Cristobal Corredor R et al.

  • With a syringe take three samples of 5 mL of blood intravenously.bg
  • Collect each of the samples in a tube of 15mL
    *Note: Do not mix in order to avoid antigen blood's coagulate
  • Add 0.5mL of EDTA at 10% pH 7.2 per sample.
  • Centrifuge at 1000 rpm for 5 min. at 12-15°C.
  • Centrifuge at 2500 rpm for 25 min. at 12-15°C.
  • Gently remove the interphase and place into a new microtube of 1.5 mL and proceed to the RNA Isolation Procedure

Isolation of mononuclear cells by density gradient separation
(McCoy, J.P., 1998. Handling, storage and preparation of human blood cells. Curr. Protocol Cytom. 5:5.1.1-5.1.13

  • With a sterile pipet, place the Ficoll-Hypaque solution into a 50-ml conical centrifuge tube, using 2 mL of Ficoll-Hypaque per 1mL of blood. (The volume of Ficoll-Hypaque may vary with brand used. Consult manufacturer's recommendations.)
  • Mix anticoagulated blood with an equal volume of PBS.
  • Slowly layer the diluted blood over the Ficoll-Hypaque solution by gently pipetting the diluted blood down the side of the tube containing the Ficoll-Hypaque.
  • Centrifuge 40 min at 400 x g, 22ºC, with no brake.
  • Using a sterile Pasteur pipet, carefully remove the mononuclear cells, located at the interface between the plasma (upper layer) and the Ficoll-Hypaque (bottom).
  • Transfer the aspirated mononuclear cells to a 15-ml conical tube. Add 10 ml PBS or tissue culture medium and mix thoroughly. Centrifuge 10 min at 400 x g, 4ºC.
  • Discard the supernatant and repeat wash with PBS or tissue culture medium as needed.

RNA Isolation Procedure with TRIzol
Preparing Samples

Homogenizing samples

  1. Determine your sample type, and perform homogenization at room temperature according to the table below. The sample volume should not exceed 10% of the volume of TRIzol® Reagent used for homogenization. Be sure to use the indicated amount of TRIzol® Reagent, because an insufficient volume can result in DNA contamination of isolated RNA.

Phase separation

  1. Incubate the homogenized sample (see Homogenizing samples) for 5 minutes at room temperature to permit complete dissociation of the nucleoprotein complex.
  2. Add 0.2 mL of chloroform per 1 mL of TRIzol® Reagent used for homogenization. Cap the tube securely.
  3. Shake tube vigorously by hand for 15 seconds.
  4. Incubate for 2–3 minutes at room temperature.
  5. Centrifuge the sample at 12,000 × g for 15 minutes at 4°C.
    Note: The mixture separates into a lower red phenolchloroform phase, an interphase, and a colorless upper aqueous phase. RNA remains exclusively in the aqueous phase. The upper aqueous phase is ~50% of the total volume.
  6. Remove the aqueous phase of the sample by angling the tube at 45° and pipetting the solution out. Avoid drawing any of the interphase or organic layer into the pipette when removing the aqueous phase.
  7. Place the aqueous phase into a new tube and proceed to the RNA Isolation Procedure.
  8. Save the interphase and organic phenol-chloroform phase if isolation of DNA or protein is desired. The organic phase can be stored at 4°C overnight.

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