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Fungal Midi DNA Kit Protocol for Fresh/Frozen Specimens

互联网2013-11-13

498

实验试剂

1. Equilibrate sterile dH₂ O water or DNA Elution Buffer at 65°C.

2. Isopropyl alcohol (isopropanol)

3. Absolute (96%-100%) ethanol

4. RNase A stock solution at 20 mg/mL

实验设备

1. Centrifuge capable of at least 8,000 x g

2. Nuclease-free 15 mL or 20 mL high speed Carbonate Thick wall centrifuge tubes

3. Waterbath equilibrated to 65°C

实验步骤

1. Collect ground Fungal tissue (start with 500 mg) in a 15 mL tube and immediately add 3mL Buffer FG 1 and 20 ul RNase A (20mg/mL). Vortex vigorously to mix the sample. Make sure to disperse all clumps. DNA cannot be effectively extracted from clumped tissue.

2. Incubate at 65°C for 30-60 min. Mix sample twice during incubation by inverting tube.

3. Add 700 ul Buffer FG 2 and vortex to mix. Incubate on ice for 10 minutes. Centrifuge at $8,000 x g for 10 min.

4. Carefully transfer supernatant into a new 15 mL centrifuge tube making sure not to loosen the pellet. Measure the volume of the sample and add 1.5 volume of prepared FG3/ethanol mixture (see instruction in Before Starting). Vortex the sample to mix throughly.

5. Apply the entire sample (including any precipitate that may have formed) to a HiBind^® DNA Midi-column placed in a 15 mL collection tube (supplied) . Centrifuge the column at 8,000 x g for 5 min to bind DNA. Discard the flow-through liquid and reuse the collection tube.

6. Place column to a same collection tube and wash by adding 3.5 mL DNA Wash Buffer diluted with absolute (96%-100%) ethanol. Centrifuge at 8,000 x g for 5 min and discard the flow-through liquid. Reuse the collection tube in Step 7 below.

NOTE: DNA Wash Buffer Concentrate must be diluted with absolute (96%-100%) ethanol prior to use. Follow directions on label.

7. Repeat wash step with an additional 3.5 mL DNA Wash Buffer. Centrifuge at 8,000 x g for 5 min. Discard flow-through and reuse 15 mL collection tube in step 8.

8. Centrifuge empty column 15 min at 8000 x g to dry the columns. This step is critical for removing residual ethanol that may otherwise be eluted with DNA and interfere with downstream applications.

9. Transfer column to another clean 15 mL collection tube (not supplied with this kit). Apply 500 ul Elution Buffer (or sterile deionized water) pre-warmed to 65°C and incubate at room temperature for 3 min. Centrifuge at 8,000 x g for 10 min to elute DNA. Smaller volumes will significantly increase DNA concentration but give lower yields. Use of more than 2 mL of buffer for elution is not recommended.

10. Repeat Step 9 with an additional 500 ul of Elution Buffer. This may be performed using another 15 mL collection tube (not supplied) to maintain a higher DNA concentration in the first eluate.

TIP: To increase DNA concentration add buffer and incubate the column at 60°C- 70°C for 10 min before elution.

Total DNA yields vary depending on type and quantity of sample. Typically, 50-250 ug DNA with a A260/A280 ratio of 1.7-1.9 can be isolated using 250 mg dried tissue.

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