Protocal Modified STRATAGENE DNA Extraction Method using
Thielaviopsis basicolaAnabel (Sep,2010)
Grow fungus in liquid culture:
- ½ strength potato dextrose broth (PDB) in sterile Petri dishes (2 x 7 mm plugs in approx. 20 ml PDB)
- leave at room temp. for 3-4 days
- (in fume hood) remove plugs
- pour fungal suspension into clean 50 ml plastic centrifuge tubes, balance with wash buffer (0.1 M NaCl)
- cap and spin at 9.5 K rpm for 10 minutes at room temp.
- carefully discard supernatant
- add 10 ml wash buffer to pellet & vortex gently
- cap and spin at 5 K rpm for 5 minutes at room temp.
- carefully discard supernatant
- add 10 ml of Solution 2 (50 mM Tris-HCl pH 8, 20 mM EDTA, 2 % SDS)
- homogenize with Dounce homogenizer to break up pellet into tiny particles (SDS will foam and give a white color)
- add proteinase K to final concentration of 200 mg/ml & vortex gently
- incubate at 55°C for 1-2 H with shaking
- chill on ice for a few minutes
- transfer supernatant to sterile Corex tubes using wide bore pipets
- add cold 95% EtOH & invert to mix
- precipitate at -20°C for a minimum of 2 H (or overnight)
- spin at 5 K rpm for 5 minutes
- wash pellet with 70% EtOH
- resuspend in TE buffer
N.B. – Run 0.7% agarose gel at this point if desired, otherwise proceed
with RNase TX and then run gel
RNase TX:
- 300 ml DNA
- 125 ml sd H2O
- 50 ml 3 M NaCl
- 25 ml RNase
- Combine DNA (in TE) with 3 M NaCl and RNase
- Incubate at 30°C for 1 H
- Add 0.8 vol isopropanol
- Invert to mix (at this point DNA strands should be readily visible)
- Spin for 5 minutes in microcentrifuge
- Wash pellet with 70 % EtOH
- Resuspend pellet in TE buffer
STRATAGENE DNA extraction (modified): Solutions
Stock Solutions:
Wash solution 0.1 M NaCl 4.38 g/750 ml sd H2O
Sat’d NaCl 6.0 M NaCl 87.66 g/250 ml sd H2O
Solution 2 50 mM Tris-HCl pH 8 75 ml 0.5 M stock
20 mM EDTA 30 ml 0.5 M stock
2% SDS 15 g
* to 750 ml sd H2O
1 Kb Ladder: Stock 250 mg/300 ml
20 ml stock in 480 ml 1 X loading buffer (=25 fold dilution)
0.2 mg/5ml loaded on gel
Tris-HCl 0.5 M 39.39 g in 500 ml H2O, ph 8.0 (using NaOH)
EDTA 0.5 M 46.53 g in 250 ml H2O, pH 8.0 (using NaOH pellets)
20% SDS 30 g in 150 ml, heat at 65°C for 1 H
DNase I TX
Assay buffer: 40 mM Tris-HCl pH 7.5
6 mM MgCl2
* Prepare as 10X stock
- 7.5 units in 20 ml final volume + 1 mg DNA
- Incubate at 37°C for 10 minutes
- Chill on ice
- Add 1 mL loading buffer to aliquot
RNase A TX
- Dissolve RNA in TE to conc. Of 1 mg/ml
- Boil for 10-30 minutes
- Cool to room temp.
- Aliquot
25 mg/ml as final concentration in 0.03 M NaCl
Incubate at 30°C for 1 H
Chill
Precipitate with isopropanol (0.8 volumes) or with NaOAc (1/10th volume)
and 95% EtOH
Incubate at 30°C for 1 H