User:Raf Aerts/Notebook/Open Coffee/2008/09/25

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Genomic DNA purification with NucleoSpin® Plant C1/C0 250

Standard protocol for isolation of genomic DNA from plants using a NucleoSpin® Plant isolation kit with buffer C1 (CTAB) or buffer C0 (detergents). Tested on Coffea arabica by RA.

Materials

  • NucleoSpin® Plant (Macherey-Nagel, Protocol June 2007/Rev. 06) Catalog # 740570.250 (250 samples)
  • EtOH 96-100%
  • Centrifuge (with a capacity of 24 samples)
  • Heatable water bassin (45-70°C)
  • 1.5 mL tubes
  • 2.0 mL tubes
  • Finnpipette 10-75 μL and 200-1500 μL
  • 3-4 tube racks
  • 1 floating tube rack
  • Stabilo OH smooth surface and NN Sharpie

Sample pretreatment

  1. Collect fresh leaf material
  2. Store leaf samples in paper bags (1 leaf/bag)
  3. Store paper bags in plastic zip-lock container with silicagel drying perls (CASP # 7150)
  4. Lyophilize samples
  5. Homogenize and powder samples
    1. Put 20 mg lyophilized (dry weight) leaf tissue (not vein material!) in a 2 ml tube (flat bottom)
    2. Add 3 pearl beads
    3. Ground to fine powder for 2 min at 30 rpm
    4. Check and repeat last step if necessary

Safety instructions

  1. Wear gloves and goggles
    1. C2: Harmful if swallowed, irritating to skin and eyes
    2. CW: Flammable, harmful if swallowed, irritating to eyes and skin
    3. RNase A: May cause sensitization by inhalation and skin contact

Preparation of working solutions

  1. Buffer C4: Transfer total content of C2 to C4 and mix
    1. 5 min incubation at 45°C recommended
    2. Store at room temperature (20-25 °C), 1 year
  2. Buffer C5: Add 200 ml EtOH (96-100%) to C5 (50 ml)
    1. Store at room temperature (20-25 °C), 1 year
  3. RNase A: 1500 μL H2O(distil) to each vial RNase A
    1. Store at 4 °C, 3 months
  4. Buffer C0: Add C to powder C0 and mix well
    1. Store at room temperature (20-25 °C), 1 year

Procedure

  1. Homogenize sample
    1. Preheat buffer C0 for 10 min at 45°C and mix well
    2. Remove beads from grinding tubes (recycle beads) and arrange tubes in rack
  2. Cell lysis
    1. Set water temperature to 60°C
    2. Add 400 μL buffer C1 (CTAB method) OR C0 (detergent-based method)
    3. Add 10 μL RNase A (yellow tips)
    4. Vortex thoroughly
    5. Arrange tubes in floating tube rack
    6. Incubate suspension for 30 min at 60 °C
    7. Prepare steps 3-8
      1. Label two sets of 1.5 ml tubes
      2. Arrange tubes in two racks (rack A and final rack)
      3. Arrange another rack (rack B) with NucleoSpin® Filter columns (violet ring) loaded onto 2 ml collecting tubes
      4. Arrange another rack (rack C) with NucleoSpin® Plant columns (grey ring) loaded onto 2 ml collecting tubes
  3. Filtration and clarification of lysate
    1. Remove tubes from water bad and set water temperature to 70 °C
    2. Load lysate onto filter columns (violet ring) (from rack B; use pipette; load directly into centrifuge; disgard grounding tubes)
    3. Centrifuge for 5 min at 11000 x g (rcf)
    4. Premix C4 and EtOH
      1. Mix (number of samples + 2) * 300 μL C4 and (number of samples + 2) * 200 μL EtOH in sterile container
      2. For a full centrifuge (24 samples): 7800 μL C4 + 5200 μL EtOH
    5. Collect 300 μL of the flow-through and transfer to new 1.5 μL tube (rack A); disgard filter and collecting tube
    6. If you need to do something else that cannot wait, now is the moment (rack A can be stored in refrigerator for some hours - no more breaks after this step)
    7. Preheat buffer CE to 70°C
  4. Adjust DNA binding conditions
    1. Add 500 μL premixed C4-EtOH (300 μL C4 + 200 μL EtOH)
    2. Mix by inverting 2-4 times
  5. Bind DNA
    1. Load sample onto NucleoSpin® Plant column (grey ring) (use pipette; rack C)
    2. Centrifuge for 1 min at 11000 x g (rcf)
    3. Repeat 1 min/11000 x g centrifuge steps until all of the lysate has passed through
    4. Discard flow-through, keep tube and column (column has the DNA)
  6. Wash silica membrane
    1. First wash
      1. Add 400 μL buffer CW to the column
      2. Centrifuge for 1 min at 11000 x g (rcf)
      3. Discard flow-through
    2. Second wash
      1. Add 700 μL buffer C5 to the column (beware: column is quite full - spat risk)
      2. Centrifuge for 1 min at 11000 x g (rcf)
      3. Discard flow-through
  7. Wash and dry silica membrane
    1. Third wash
      1. Add 200 μL buffer C5 to the column
      2. Centrifuge for 2 min at 11000 x g (rcf)
      3. Column should be completely dry
  8. Elute pure DNA
    1. Place column on final 1.5 ml tube (final rack)
    2. Pipette 75 μL elution buffer CE (preheated! to 70 °C) onto column (yellow tips)
      1. If eluted DNA does not reach required concentration, alternatively elute twice by using 50 μL elution buffer CE
    3. Incubate at room temperature for (at least) 5 min
    4. Centrifuge for 1 min at 11000 x g (rcf) (take care with lids here)
    5. Store final rack safely





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