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
- Collect fresh leaf material
- Store leaf samples in paper bags (1 leaf/bag)
- Store paper bags in plastic zip-lock container with silicagel drying perls (CASP # 7150)
- Lyophilize samples
- Homogenize and powder samples
- Put 20 mg lyophilized (dry weight) leaf tissue (not vein material!) in a 2 ml tube (flat bottom)
- Add 3 pearl beads
- Ground to fine powder for 2 min at 30 rpm
- Check and repeat last step if necessary
Safety instructions
- Wear gloves and goggles
- C2: Harmful if swallowed, irritating to skin and eyes
- CW: Flammable, harmful if swallowed, irritating to eyes and skin
- RNase A: May cause sensitization by inhalation and skin contact
Preparation of working solutions
- Buffer C4: Transfer total content of C2 to C4 and mix
- 5 min incubation at 45°C recommended
- Store at room temperature (20-25 °C), 1 year
- Buffer C5: Add 200 ml EtOH (96-100%) to C5 (50 ml)
- Store at room temperature (20-25 °C), 1 year
- RNase A: 1500 μL H2O(distil) to each vial RNase A
- Store at 4 °C, 3 months
- Buffer C0: Add C to powder C0 and mix well
- Store at room temperature (20-25 °C), 1 year
Procedure
- Homogenize sample
- Preheat buffer C0 for 10 min at 45°C and mix well
- Remove beads from grinding tubes (recycle beads) and arrange tubes in rack
- Cell lysis
- Set water temperature to 60°C
- Add 400 μL buffer C1 (CTAB method) OR C0 (detergent-based method)
- Add 10 μL RNase A (yellow tips)
- Vortex thoroughly
- Arrange tubes in floating tube rack
- Incubate suspension for 30 min at 60 °C
- Prepare steps 3-8
- Label two sets of 1.5 ml tubes
- Arrange tubes in two racks (rack A and final rack)
- Arrange another rack (rack B) with NucleoSpin® Filter columns (violet ring) loaded onto 2 ml collecting tubes
- Arrange another rack (rack C) with NucleoSpin® Plant columns (grey ring) loaded onto 2 ml collecting tubes
- Filtration and clarification of lysate
- Remove tubes from water bad and set water temperature to 70 °C
- Load lysate onto filter columns (violet ring) (from rack B; use pipette; load directly into centrifuge; disgard grounding tubes)
- Centrifuge for 5 min at 11000 x g (rcf)
- Premix C4 and EtOH
- Mix (number of samples + 2) * 300 μL C4 and (number of samples + 2) * 200 μL EtOH in sterile container
- For a full centrifuge (24 samples): 7800 μL C4 + 5200 μL EtOH
- Collect 300 μL of the flow-through and transfer to new 1.5 μL tube (rack A); disgard filter and collecting tube
- 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)
- Preheat buffer CE to 70°C
- Adjust DNA binding conditions
- Add 500 μL premixed C4-EtOH (300 μL C4 + 200 μL EtOH)
- Mix by inverting 2-4 times
- Bind DNA
- Load sample onto NucleoSpin® Plant column (grey ring) (use pipette; rack C)
- Centrifuge for 1 min at 11000 x g (rcf)
- Repeat 1 min/11000 x g centrifuge steps until all of the lysate has passed through
- Discard flow-through, keep tube and column (column has the DNA)
- Wash silica membrane
- First wash
- Add 400 μL buffer CW to the column
- Centrifuge for 1 min at 11000 x g (rcf)
- Discard flow-through
- Second wash
- Add 700 μL buffer C5 to the column (beware: column is quite full - spat risk)
- Centrifuge for 1 min at 11000 x g (rcf)
- Discard flow-through
- Wash and dry silica membrane
- Third wash
- Add 200 μL buffer C5 to the column
- Centrifuge for 2 min at 11000 x g (rcf)
- Column should be completely dry
- Elute pure DNA
- Place column on final 1.5 ml tube (final rack)
- Pipette 75 μL elution buffer CE (preheated! to 70 °C) onto column (yellow tips)
- If eluted DNA does not reach required concentration, alternatively elute twice by using 50 μL elution buffer CE
- Incubate at room temperature for (at least) 5 min
- Centrifuge for 1 min at 11000 x g (rcf) (take care with lids here)
- Store final rack safely
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