FungalGenomicDNAExtraction

實驗概要

This procedure does not require phenol extraction. The DNA is pure enough for restriction digests, PCR and genomic library construction.
High throughput of many fungal isolates can be achieved by growing axenic cultures in either (a) 1.5mL microfuge tubes, half full with liquid media (500uL), with a hole punched through the top for aeration; or if greater volumes are required, (b) peteri plates containing 10mL liquid media.In both cases, media is decanted and several water washes done to removemedia carbohydrates.

實驗步驟

1. If using petri plates, the 3-7 day culture is then diced up, using asterile scalpel, and small pieces (ca. 50-100mg) are placed in microfuge tubes containing sterile sand (ca. 100mg) and 500uL of extraction buffer. If using microfuge tubes, simply add the sand and extraction buffer:Using a Kontes micro-homogenizer with sterilized tips (Fisher Scientific Cat. # K749540-0000) samples are ground into a slurry and incubated for 60 min. at 60C.

    (tubes can be stored in this extraction buffer at minus 20C for greater than a year)

• 100mM Tris, pH8.0

• 10mM EDTA

• 2% SDS

• 100ug/mL Proteinase K

• 1% B-mercaptoethanol

2. Using a Kontes micro-homogenizer with sterilized tips (Fisher Scientific Cat. # K749540-0000) samples are ground into a slurry and incubated for 60 min. at 60C.

3. Salt concentration is adjusted to 1.4M with 5M NaCl, 1/10 vol. of 10% CTAB added and samples incubated a further 10 min. at 65C.

4. Add 1 vol. chloroform:isoamyl alcohol, gently emulsify by inversion, incubate  at 0C for 30 min. Spin 10 min. at 4C at rpm max. Transfer top phase to fresh 1.5mL microfuge tube, add 1/2 vol. 5M NH4OAc, mix gently, ice for 60 min.; spin at 4C at rpm max.

5. Transfer supernatant to fresh tube (add stock RNase 10mg/mL to a final concentration of 0.02ug/uL) add 0.55 vol. isopropanol to precipitate the DNA. Spin immediately 5-10 min. at rpm max. Aspirate off supernatant, wash DNA pellet twice with 70% ETOH, air dry pellet 20 min. and resuspend in 50uL  TE buffer. Incubate 4C overnight.

6. Quantify yield with the Hoefer DyNA Quant 200 fluorometer.

This procedure does not require phenol extraction. The B-mercaptoethanol can be omitted from the extraction buffer for safety, but yields will be slightly lower. The DNA is pure enough for restriction digests, PCR and genomic library construction.