DNA extraction

John Pitkin jpitkin at MSU.EDU
Thu Nov 16 12:32:07 EST 1995


We use a modified version of a plant CTAB DNA extraction protocol for
extracting DNA from Cochliobolus carbonum.  Jenifer Murphy in our lab has
used this method to successfully isolate DNA from C. hetrostrophus,
Aternaria, and Fusarium.  The key elements in this prep are (1) the use of
young lyophilized mycelial mats....young mats (4 days growth for C.
carbonum)...yield less contaminating carbohydrates and other misc. junk
(2) lots of proteinase K in the extraction buffer to kill DNases (final
=0.3mg/ml).

Protocol

-  place 0.2- 0.5 g (dry wt) lyophilized pad in a 5o ml disposable
centrifuge tube, break up the pad  wtih a spatula or glass rod, add c. 5 ml
3mm glass beads and powder the pad by brief shaking.
- add 10 ml (for a 0.5 g pad) of CTAB extraction buffer(see recipe, below),
gently mix to wet all the powdered pad
-place in 65 C water bath for 30 min
-cool, add equal vol. ChCl3:IAA (24:1)
-mix, centrifuge in table top fuge 10 min at full speed
-transfer aqueous supernatant to a new tube
-add an equal volume of isopropanol
-High mw DNA should spool out upon mixing...spool out the DNA with a glass
rod or hook, pour out the remaining supernatant
- rinse the spooled DNA with 70% ethanol
-air dry, add 1- 5 ml TE containing 20 ug/ ml Rnase A. To resuspend the
samples, place in 65 C bath, or allow pellets to resuspend overnight at 4 C

Notes- if the spooled DNA is discolored or has contaminating mycelial
debris, phenol/chlorofrom extract and precipitate w/ ethanol.
-This protocol can be scaled down using 0.1g pad in a 2ml eppendorf tube
- For Southerns we routinely cut 50- 75 ul (2-4 ug) of a standard DNA prep
in 200 ul rxn volumes, ETOH ppt, and resuspend in 30 ul.
-even after digestion the resuspended DNA can be very viscous at room temp;
To load a Southern we keep the samples in a 50- 60 C heat block while
loading to keep the samples at a lower viscosity.

CTAB extraction buffer: O.1M Tris, pH 7.5, 1% CTAB (mixed hexadecyl
trimethyl ammonium bromide), 0.7M NaCl, 10mM EDTA, 1% 2-mercaptoethanol.
Add proteinase K to a final concentration of 0.3 mg/ml prior to use.  Less
prot K may be acceptable for different fungi, and in fact we haven't
determined if we can use less...this conc. was calibrated for a different C.
carbonum DNA extraction buffer.

Reference:Saghai-Maroof MA, Soliman KM, Jorgensen RA, Allard RW (1984)
P.N.A.S. USA 81:8014-8018
John Pitkin
MSU-DOE Plant Research Laboratory
Michigan State University
E. Lansing, MI 48824
Tel= (517)353-4886
e-mail= JPITKIN at MSU.EDU




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