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Poplar Genomic DNA Extraction Protocol Rick Meilan 21 October 2004 Dice ~100 mg leaf tissue with a single-edge razor and transfer to 2.0-mL Eppendorf screw-top tube Add 1 mL of Modified Black Walnut HTIRC Extraction Buffer1 Add sufficient β–mercaptoethanol to achieve 2% (20 μL) Add ceramic bead Shake 3X for 40 sec. each in Savant FastPrep (model FP120)2; sample on ice between each Invert overnight at 60 ºC3 The next morning, withdraw 1.0 mL liquid and transfer it to a clean 1.5-mL Eppendorf tube Centrifuge samples to pellet debris Transfer 400 μL of the supernatant to a fresh 1.5-mL Eppendorf tube Add 4 μL of RNase stock solution (100 mg/mL) from the Qiagen kit (DNeasy® Plant Mini Kit) Perform steps 3-13 (skip steps 1 and 2) of protocol that came with the DNeasy kit (Handbook for DNA isolation from plant tissue), beginning on p. 15. 1 A stock solution of this is made up and stored at 4 ºC in PFEN 141; it must be heated in a water bath at least 10 minutes before use to ensure all components go into solution. See Rob Robichaud concerning where the stock is stored and for permission to use it. 2 This piece of equipment is in PFEN 141 on a bench in the northwest corner of the room. Contact Lab Manager (Marcia Kremer) for instruction on how to use it. 3 There is a device housed in an incubator in PFEN 141 (against south wall, to east of the laminar flow hood) that will invert tubes automatically. The incubator has to be turned on in advance, so it can warm up. Again, ask the Lab Manager for instructions and permission to use this equipment.
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