FungalMidiDNAKitOptionalprotocol

實驗概要

This protocol is designed for isolation of genomic DNA from fresh, frozen, or dried specimens from fungal samples contains higher phenolic material and polysaccharides which could cause lower yield or clogging of column with standard protocol.

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

Dry Samples - use a maximum of 150 mg ground tissue

Fresh Samples - use a maximum of 500 mg fresh/frozen ground tissue

1. Collect ground sample in a centrifuge tube and add 4mL Buffer FG1 and 20 ul RNase (20 mg/mL). Vortex vigorously to mix

2. Incubate at 65°C for at least 25 min. Mix sample once during incubation by inverting tube.

3. Add 750 ul Buffer FG2 and vortex to mix. Incubate on ice for 5 minutes. Centrifuge at 8,000-10,000 x g for 10 min.

4. Carefully transfer cleared lysate to a new high speed centrifuge tube making sure not to disturb the pellet or transfer any debris. Add 0.7 volume isopropanol and vortex to precipitate DNA. This step will remove much of the polysaccharides and improve spin-column performance by increasing DNA binding capacity (and hence yield) in the steps that follow. No incubation is required after addition of isopropanol.

TIP: In most cases 2.5 mL supernatant can easily be removed. This will require 1.75mL (0.7 volume) isopropanol. Note that depending on the sample type, the volume of supernatant may vary. After transferring to a fresh tube, measure the volume and add the correct amount of isopropanol.

5. Immediately centrifuge at 8,000 x g for 15 min to pellet DNA. Longer centrifugation does not improve yields.

6. Carefully aspirate or decant the supernatant and discard making sure not to loosen the DNA pellet. Invert the centrifuge tube and place on a paper towel for 5 min to allow residual liquid to drain. It is not necessary to dry the DNA pellet.

7. Add 1.5 mL of sterile deionized water pre-heated to 65°C and vortex to resuspend the pellet. A brief incubation at 65°C may be necessary to effectively dissolve the DNA. Add 20 ul RNase (20 mg/mL) and mix. No additional incubation is required for RNase treatment.

TIP: While incubating at 65°C to dissolve the DNA, label and place the required number of HiBind^? DNA columns in 15 mL collection tubes (supplied).

8. Adjust binding conditions of the sample by adding 750 ul Buffer FG3 followed by 1.5 absolute ethanol and vortex to obtain a homogeneous mixture. A precipitate may form upon addition of ethanol; it will not interfere with DNA isolation. A precipitate may form upon addition of ethanol; it will not interfere with DNA isolation. Break the precipitation by pipetting up and down 10-20 times to obtain a homogeneous mixture.

9. Apply the entire sample (including any precipitate that may have formed) to a HiBind^? Midi-spin DNA column placed in a 15 mL collection tube (supplied). Centrifuge the column at 8,000 x g for 10 min to bind DNA. Discard the flow-through liquid and reuse the collection tube.

10. Reassemble column to same collection tube and wash by adding 3.5 mL Wash Buffer diluted with absolute (96%-100%) ethanol. Centrifuge at 8,000 x g for 10 min and discard the flow-through liquid. Reuse the collection tube in Step 11 below.

11. Repeat wash step with an additional 3.5 mL Wash Buffer. Centrifuge at 8,000 x g for 5 min. Discard flow-through and reuse 15 mL collection tube in Step 12.

12. Centrifuge empty column 10 min at 8,000 x g to dry the column. This step is critical for removing residual ethanol that may otherwise be eluted with DNA and interfere with downstream applications.

13. Transfer column to a clean 15 mL tube (not supplied with this kit). Apply 500 ul DNA Elution Buffer (or sterile deionized water) pre-warmed to 65°C and incubate at room temperature for 1 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 2mL of buffer for elution is not recommended.

14. Repeat Step 13 with an additional 0.5 mL of Elution Buffer. This may be performed using another 15 mL tube 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 5 min before elution.

15. Store the eluted DNA at -20°C