CTAB Prep for Magnaporthe grisea (Shaking Culture)
1. Start a culture of the isolate in 150ml of CM media in a 250ml flask with a 1" diameter agar plug of actively growing Magnaporthe (Penicillin/Streptomycin) can be added (to 150 ug/ml) to media to avoid problems with bacterial contamination).
-Culture for 48 hours, 125 rpm on shaker in growth room.
2. Filter through 2 layers of miracloth and blot dry with paper towels, removing as much liquid as possible.
- proceed to step 3 or wrap in foil and freeze in liquid nitrogen and store at -70oC.
3. Place the sample in a mortar with liquid nitrogen and quickly grind the sample to a fine powder (mycelia should powder easily).
4. Transfer powder to a sterile Oakridge tube containing 5mL 2X CTAB buffer at 65C and incubate for 20 min@65o, inverting the tube every 10 minutes to further break up fungal material (this step inactivates cellular proteins and allows the CTAB to complex with contaminating carbohydrates).
5. Add 5 ml CIA {Chloroform/pentanol (24:1)} to the tubes and let stand uncapped in the fume hood for 1 minute.
Replace caps and gently mix on shaking platform for 20 minutes.
centrifuge 10 min@10 K @4oC (in swinging bucket rotor).
6. Carefully remove aqueous (top) phase (be sure not to take any of the organic phase or cellular debris) and repeat Chloroform/pentanol extraction.
7. After the second Chloroform/pentanol wash, remove aqueous phase and add to 5mL isopropanol in an Oakridge tube, to precipitate nucleic acids.
at this step it is even more important not to pick up any of the CIA phase, so be very careful to leave behind a few mm of the upper phase.
invert tube gently a few times to mix the sample and let sit for 5 minutes on ice to allow complete precipitation of the nucleic acids (sometimes you can see the DNA at this point. If you can't, don't panic).
8. Centrifuge at 10K/10 min.4oC
Decant supernatant and drain tubes on a paper towel for about 15 min (at this point there should be a white pellet at the bottom of the tube).
9. Resuspend the pellet in 500 ul 1X TE.
after the pellet is no longer visible transfer the sample to an eppendorf tube. Phenol/chloroform (1:1) extraction. Reprecipitate the nucleic acids with 3 M NaOAc (1/10th vol.) and 2 vol. of ethanol.
incubate at -20oC for 10 minutes and centrifuge at 13,000 rpm in microfuge for 20 min.
wash pellets once with 70% ethanol and spin in microfuge for 5 min.
drain and dry pellets on paper towel.
10. Resuspend pellets in 25-100 ul of TE+RNase (less if the pellets are small).
note: clean DNA pellets will resuspend completely at room temp. in only a few hours (do not pipette solution up & down as this will tend to shear the DNA).
-samples may appear to be cloudy when resuspended, but this does not seem to interfere with restriction digestion.
CTAB Extraction Solutions
CTAB Buffer[500ml]
CTAB2%10g
Trisma Base100mM6.06g
EDTA10mM1.46g
NaCl0.7M20.5g
note: CTAB is Hexadecyltrimethylammonium Bromide (Sigma # H-5882)
CIA24 : 1 (V/V) Chloroform : Pentanol