Another DNA extraction protocol

Harvey E. Ballard, Jr. hballard at STUDENTS.WISC.EDU
Tue Feb 18 18:41:45 CST 1997


Dr. Eddie asked about another DNA extraction protocol that was perhaps less
messy or less time-consuming, and at least as effective in ameliorating
problems with polysaccharides.  The Violaceae I work with, especially
Viola, often have terrible "snot" in the leaf tissue.  Using 6% CTAB in the
Doyle & Doyle procedure works best with very mucilaginous taxa of all
kinds.

Recently, however, in order to take full advantage of small amounts of
herbarium specimen leaf tissue as well as fresh, frozen and silica gel
dried tissue, I've produced a "hybrid" mini-extraction protocol for
Eppendorf tubes that uses the D&D approach for much of it but with an SDS
buffer (Edwards, K., Johnstone, C., & Thompson, C. 1991. Nucleic Acids
Research 19:1349).  Note the Notes at the bottom of the protocol about a
new modification that makes it even easier and quicker.  I hope it works
for others!

My best,
Harvey Ballard

=+=+=+=+=+=+=+=+=+=
MODIFIED MINI-EXTRACTION PROTOCOL
Harvey Ballard, 16 November 1995

(Procedure takes ca. 3 hours from start to finish for 18 samples for the
complete procedure below; about 1-1/2 using only two chloroform-isoamyl
alcohol precipitations and the 70% ethanol rinse at the end.)

1  Label 2 sets of 1.5 ml Eppendorf tubes for your samples

2  Place ca. 1 lid-worth (approximately a paper punch-sized piece) of leaf
tissue for silica-gel or fresh material, 2 lids-worth of herbarium material
in one set of tubes; coarse-grind leaf tissue with blunt-nosed forceps or
another slender tool with broad end

3  Add 400 microliters Mini-Extraction Buffer to each tube; vortex at
moderate speed briefly to mix well; let tubes sit at room temperature or in
hot-water bath for 5 min to hydrate leaf tissue

4  Use mini-pestles to fine-grind tissue in each tube; add another 200
microliters extraction buffer; vortex at moderate speed briefly to mix
well; soak tubes in hot-water bath for 5 min and grind once more with
pestle

5  Under hood: add 500 microliters 24:1 chloroform-isoamyl alcohol mixture
under hood, invert twice carefully and lift cap to remove initial pressure,
then shake tube vigorously to form emulsion, finally lifting the cap again
(slowly!) to expel pressure; centrifuge at 13,000 rpm for 5 min

6  Transfer 350-400 microliters of clear upper aqueous phase (avoid
particles!) to second set of tubes under hood; add 300 microliters
isopropanol, invert several times to mix; freeze for 20 min in -80 degree
ultra-cold freezer to precipitate DNA

7  Centrifuge at 13,000 rpm for 5 min; pour off supernatant into waste
beaker at hand; add 100 microliter TE buffer and vortex at moderate speed
briefly to help dissolve (can heat for 5 min in hot-water bath to help)

8  Add 50 microliters 7.5 M ammonium acetate and 300 microliters 95%
ethanol to tubes; invert several times to mix; freeze for 20 min in
ultra-cold freezer

9  Centrifuge  at 13,000 rpm for 5 min; pour off supernatant into waste
beaker at hand; add 100 microliters TE buffer and vortex at moderate speed
briefly to help dissolve

10  Add 10 microliters 2.5 M sodium acetate and 200 microliters 95% ethanol
to tubes; invert several times to mix; freeze for 2-5 min in ultra-cold
freezer

11  Centrifuge  at 13,000 rpm for 5 min; pour off supernatant into waste
beaker at hand

12  Add 100 microliters 70% ethanol; let tubes sit undisturbed for 5 min;
gently pour off ethanol into waste beaker, making sure that you don't lose
the pellet adhering to the side of the tube

13  Tip each tube upside down and gently tap onto paper towel to remove
excess ethanol from tube walls (be careful not to dislodge pellet!

14  Put rack of tubes in vacuum pump for 10-15 min or sit out at room
temperature for 1-2 hrs to permit complete evaporation of ethanol.

15  Resuspend in 100 microliters TE buffer.

---
Mini-Extraction (SDS) Buffer:
100 ml 2M Tris buffer stock
14.61 g NaCl
50 ml 0.5M EDTA buffer stock
5 g SDS
bring to 1 liter volume with distilled water


TE Buffer:
1.21 g Tris
0.37 g EDTA
bring to 1 l volume with distilled water
pH to 7.4 with Hcl


---
Notes:
1) While I don't use liquid nitrogen with violet or other samples that I've
extracted using this method, other folks in the lab have fashioned a little
aluminum foil funnel to fit the Eppendorf tubes, through which they pour a
small amount of N2 on the leaf tissue before grinding.

2) We are now experimenting with simply doing TWO chloroform-isoamyl
extractions, one after the other, rinsing with 70% ethanol and drying down,
then resuspending in TE, and dropping the acetate salt rinses entirely.
This looks as though it will save time and yield at least as good an
extract.

POTENTIAL MODIFICATION #1:  While I haven't bothered with
refrigerating/freezing samples for a few days at the end of step #6, folks
in the Mark Chase lab (I hear) have gotten increased yields of precipitated
DNA from a prolonged, cold "rest stop" at this point.  It might be worth
trying for recalcitrant plant groups for which older herbarium material is
extracted.

POTENTIAL MODIFICATION #2:  Scott Hodges uses the SDS buffer solution but
with the following procedure:
1) grind leaf tissue in the tube with liquid nitrogen (and a pinch of sand
with herbarium material)
2) add 400 ul buffer and 1-2 ul proteinase K (10mg/ml) and vortex
3) heat at 37oC 30 min
4) add phenol-chloroform solution, put on "wheel" until pigments move into
phenol
5) remove aqueous phase, add 1-2 ul RNAse, heat at 37oC 30 min
6) extract with chloroform-isoamyl as in step #5 in first protocol
7) add equal volume isopropanol, shake, let stand 2 min
8) centrifuge top speed 5 min
9) pour off aqueous solution, dry and resuspend in 50 ul TE or H2O



********
Harvey E. Ballard, Jr., Ph.D.
Postdoctoral Researcher, USDA, Agricultural Research Service
Horticulture Department, University of Wisconsin
1575 Linden Drive, Madison WI 53706
phone: (608) 262-0159
and
Honorary Fellow, University of Wisconsin Herbarium
Botany Department, UW
132 Birge, 430 Lincoln Drive, Madison WI 53706
phone: (608) 262-2792
fax: (608) 262-7509

AFTER 31 JULY:
Assistant Professor
Department of Environmental and Plant Biology
Porter Hall
Ohio University
Athens OH 45701
Phone: To be announced
Fax: (614) 593-1130
Email: A mystery as yet




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