E-Z 96 Fungal DNA Protocol (Centrifugation Processing)
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实验试剂
1. Equilibrate sterile dH2 O water or 10 mM Tris pH 9.0 at 65℃.
2. Isopropyl alcohol (isopropanol; absolute)
3. Absolute (96%-100%) ethanol
4. RNase A stock solution at 20 mg/mL
5. Liquid nitrogen for freezing/disrupting samples (For fresh/Frozen specimens)
实验设备
1. Laboratory centrifuge equipped with swinging-bucket rotor (for centrifugation protocol).
2. Rotor-adapter for deep-well microplate (for centrifugation processing)
3. Nuclease-free 1.5 mL or 2 mL microfuge tubes
4. Waterbath equilibrated to 65℃
5. Vacuum manifold (for vacuum manifold processing only)
实验步骤
1. Transfer the entire sample from step 8 of Section I into the E-Z 96® DNA plate. Seal the top of the DNA plate with adhesive plate film. Use a register chart to identify the positions of the samples.
Note: Do not touch the rims of the wells with pipet tips to avoid cross-contamination.
2. Place the E-Z 96® DNA plate atop a 2 mL 96 well collection plate (supplied). Connect the two plates by using plastic tape if necessary.
3. Place the plates into the centrifuge and spin at 4,000 x g for 5 min.
4. Separate the plates and discard the flow-through liquid. Re-use the collection plate.
5. Remove the adhes ive plate film and carefully add 700 ul of DNA wash Buffer to each well of the E-Z 96® DNA plate.
6. Seal the E-Z 96® DNA plate with new adhesive plate film.
7. Reassemble the E-Z 96® DNA plate with the collection plate. Centrifuge at 4,000 x g for 5 min.
8. Separate the plates and discard the flow-through liquid. Wash the DNA plate again with another 700 uL DNA wash Buffer by repeating step 5-7.
9. Remove the adhesive film, discard the flow-through and place the E-Z 96® DNA plate atop the 300uL collection plate (supplied). Centrifuge at 4,000 x g for 10 minutes.
Note: Drying the membrane at this step is very important for DNA elution in next step. The residue of the DNA wash buffer contains ethanol which will inhibit PCR and cause low yield of DNA.
10. To elute the DNA, add 100 ul of preheated (65℃) Elution Buffer to each well using a mutichannel pipet. Seal the E-Z 96® DNA plate with new adhesive film and incubate for 5 min at room temperature. Centrifuge at 4,000 x g for 5 min.
100 ul Elution Buffer is sufficient to elute up to 85% of the DNA from each well of the E-Z 96® DNA plate. A second elution step with same 100 ul elute containing DNA, reheated to 65℃, will increase yield by up to 10-15%
