FungalMidiDNAKitProtocolforFresh／FrozenSpecimens

實驗概要

This  protocol is suitable for most fresh or frozen tissue samples allowing  more efficient recovery of DNA. However, due to the tremendous variation  in water and polysaccharide content in fungi, sample size should be  limited to ≤1g.

To prepare samples collect tissue in a 30mL mortar and freeze by  dipping in liquid nitrogen with a pair of tweezers to fill the tube.  Grind the tissue using clean pestles. Alternatively, one can allow  liquid nitrogen to evaporate and then store samples at -70°C for later  use. For critical work such as PCR and cloning, pestles are best used a  single time then soaked in a dilute bleach solution immediately after  use until clean. Disposable pestles may be autoclaved several times. For  standard Southern analysis, the same pestle can be reused several times  to grind multiple tissue samples by rinsing with ethanol and carefully  wiping the surface clean between samples.

主要試劑

1. Equilibrate sterile dH₂O 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

主要設備

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

實驗步驟

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.