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
 * 6) Put 20 mg lyophilized (dry weight) leaf tissue (not vein material!) in a 2 ml tube (flat bottom)
 * 7) Add 3 pearl beads
 * 8) Ground to fine powder for 2 min at 30 rpm
 * 9) Check and repeat last step if necessary

Safety instructions

 * 1) Wear gloves and goggles
 * C2: Harmful if swallowed, irritating to skin and eyes
 * CW: Flammable, harmful if swallowed, irritating to eyes and skin
 * 1) 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
 * 2) 5 min incubation at 45°C recommended
 * 3) Store at room temperature (20-25 °C), 1 year
 * 4) Buffer C5: Add 200 ml EtOH (96-100%) to C5 (50 ml)
 * 5) Store at room temperature (20-25 °C), 1 year
 * 6) RNase A: 1500 μL H2O(distil) to each vial RNase A
 * 7) Store at 4 °C, 3 months
 * 8) Buffer C0: Add C to powder C0 and mix well
 * 9) Store at room temperature (20-25 °C), 1 year

Procedure

 * 1) Homogenize sample
 * 2) Preheat buffer C0 for 10 min at 45°C and mix well
 * 3) Remove beads from grinding tubes (recycle beads) and arrange tubes in rack
 * 4) Cell lysis
 * 5) Set water temperature to 60°C
 * 6) Add 400 μL buffer C1 (CTAB method) OR C0 (detergent-based method)
 * 7) Add 10 μL RNase A (yellow tips)
 * 8) Vortex thoroughly
 * 9) Arrange tubes in floating tube rack
 * 10) Incubate suspension for 30 min at 60 °C
 * 11) Prepare steps 3-8
 * 12) Label two sets of 1.5 ml tubes
 * 13) Arrange tubes in two racks (rack A and final rack)
 * 14) Arrange another rack (rack B) with NucleoSpin® Filter columns (violet ring) loaded onto 2 ml collecting tubes
 * 15) Arrange another rack (rack C) with NucleoSpin® Plant columns (grey ring) loaded onto 2 ml collecting tubes
 * 16) Filtration and clarification of lysate
 * 17) Remove tubes from water bad and set water temperature to 70 °C
 * 18) Load lysate onto filter columns (violet ring) (from rack B; use pipette; load directly into centrifuge; disgard grounding tubes)
 * 19) Centrifuge for 5 min at 11000 x g (rcf)
 * 20) Premix C4 and EtOH
 * 21) Mix (number of samples + 2) * 300 μL C4 and (number of samples + 2) * 200 μL EtOH in sterile container
 * 22) For a full centrifuge (24 samples): 7800 μL C4 + 5200 μL EtOH
 * 23) Collect 300 μL of the flow-through and transfer to new 1.5 μL tube (rack A); disgard filter and collecting tube
 * 24) 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)
 * 25) Preheat buffer CE to 70°C
 * 26) Adjust DNA binding conditions
 * 27) Add 500 μL premixed C4-EtOH (300 μL C4 + 200 μL EtOH)
 * 28) Mix by inverting 2-4 times
 * 29) Bind DNA
 * 30) Load sample onto NucleoSpin® Plant column (grey ring) (use pipette; rack C)
 * 31) Centrifuge for 1 min at 11000 x g (rcf)
 * 32) Repeat 1 min/11000 x g centrifuge steps until all of the lysate has passed through
 * 33) Discard flow-through, keep tube and column (column has the DNA)
 * 34) Wash silica membrane
 * 35) First wash
 * 36) Add 400 μL buffer CW to the column
 * 37) Centrifuge for 1 min at 11000 x g (rcf)
 * 38) Discard flow-through
 * 39) Second wash
 * 40) Add 700 μL buffer C5 to the column (beware: column is quite full - spat risk)
 * 41) Centrifuge for 1 min at 11000 x g (rcf)
 * 42) Discard flow-through
 * 43) Wash and dry silica membrane
 * 44) Third wash
 * 45) Add 200 μL buffer C5 to the column
 * 46) Centrifuge for 2 min at 11000 x g (rcf)
 * 47) Column should be completely dry
 * 48) Elute pure DNA
 * 49) Place column on final 1.5 ml tube (final rack)
 * 50) Pipette 75 μL elution buffer CE (preheated! to 70 °C) onto column (yellow tips)
 * 51) If eluted DNA does not reach required concentration, alternatively elute twice by using 50 μL elution buffer CE
 * 52) Incubate at room temperature for (at least) 5 min
 * 53) Centrifuge for 1 min at 11000 x g (rcf) (take care with lids here)
 * 54) Store final rack safely


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