PLANT MOLE('ULAR BIOI.

OGY REPORTER
Volume 1, Number .t, Fall 198~ pages 19-21

EXPERIMENTAL PROTOCOLS

A Plant D N A
Minipreparation:
Version II
Stephen L. D e l l a p o r t a , Jonathan W o o d , J a m e s B. H i c k s
Cold Spring Harbor l.ab.rat,~ry. Cold Sprtng Harbor. N "1" 11724

The topic of this report is rap,d m,croscale methods for ,solat,on of plant
D N A without tile use of ultracentr,fugatlon wEth CsCI. The D N A produced
,s of moderately high molecular weight and serves as a satisfactory substrate
for most restrlctum cndonucleases and is statable for genom,c blot analys,s.
In addition to the rapidity and convenience of mlmpreps which permit a large
number of samples to be processed in just a few hours, the small amount of
tissue reqmred (less than 1.0 grams) allows tbr molecular analysis of plants at
a very young stage M m , p r e p D N A y,elds from leaf tissue of most species
tested to date are typ,cally 30-100 big per gram tissue, greater than 50 kb,
and remarkably uniform from sample to sample.
The first m m l p r e p procedure we reported fi3r maize D N A isolation (Della-
porta et al , ;'*l,;tze Geneta3 Cr162162 Neu'_~letlrt. 1983) was adapted from a
procedure commonly used for }'east D N A preparatmn (Dav,s et al., 1980)
Since th,s report, numerous personal commun,cat,ons have demonstrated that
the m m , p r e p procedure or a modification thereof, can be apphed to most
plant species tested. For example, the method has been successfully used on
Ntcottana hlgl~um. N. plumklgmgidtum. N. 3)/t'eJtrt~. L)s~opertcum sp.. Amar-
,mthm sp . Gl)~me max. Petuma h.~hra&. Several modifications have been ap-
phed by these ,nvestlgators and in our own laboratory m order to extend the
appl,catmn of ram,prep procedures to other plant species. The select,on of a
particular protocol depends to a large degree on the plant spec,es used. How-
ever, the procedure reported here was selected to be statable for most situa-
tions.

19
2O Plant ~,lolecuD1r B1,,D4~3 Rep,rto"

Miniprep Procedure
1 Weigh () 5 to 75 gm of leaf tissue, quick freeze in hquid nitrogen and
grind to a fine powder ,n a 3 m. mortar and pestle Transfer powder with
liqmd nitrogen into a ~CI ml Oak Ridge tube
It is m~perative not t~J let the tissue thaw once frozen until buffer is added
and not tt) c a p t h e tubes wh~le nitrogen Is evaporating
2 Add 15 ml of Extr,tction Buffer (F.B) 100 mM Trls pH 8, 51) mbl EDTA
pH 8, 500 mM NaCI, 10 mM mcrcaptoethanol
For maxHnum D N A yields, the cells ,ire further broken by grinding the
inixture ,it a low settmg (about ~) x~lth a Polytron (Brmkmann Instruments.
Inc ) However, this step is optmnal
a, Add 1 (} ml of 2(}~'; SDS. mix thoroughly by vigorous shaking, and
mcub,ite tubes ,it 65~ for I() m m
t Add 5 0 nil 5 M potassium acetate. Shake tube vigorously and incubate
(1~ tbr 20 rain.
Most proteins ,lnd polys,icch,irides ,ire removed ,is ,l cilmplex w i t h the in-
soluble pot,iSSlUm dodecyl sulfate preciplt,ite
5 Spin tubes ,it 25,()01} X g filr 20 rain Pour supernatant t h r o u g h ,l m i r -
,lchith filter (C,tlbiochem) into ,l clean "~0 ml ttlbL" corlt,lirlin<l,~ l() ml isopro-
panol Mix and i n c u b a t e t u b e s ,it - 2 ( 1 ~ for 2~() lllln
6 Pellet DNA ,it 21).00() ~ g for 15 rain Gently pour offsupern,itant
and lightly dry pellets by inverting the tubes on paper towels tor 1() rain
v Redlssulve D N A pellets with 0 v ml of 50 mM Tris, 1(1 mM EDTA,
pH 8 Transfer the solution to ,in E p p e n d o r f t u b e Spin the tubes in a micro-
fuge tor 1() rain to remove insl>luble debris
8 TranslTer tile supcrnatant to a new Eppendorf tube and add 75 i.tl ~,M
sodium acetate and 500 hi.1 isoprop,lnol Mix well and pellet the clot of DNA
for ~,() set in ,l microfuge Wash pellet with Si)'; ethanol, dry, and redissolve
in 10() I.tl 1() mM Tris, 1 mM EDTA, pH 8
Precipitation from () a, M s o d i u l l l a c e t a t e using relatively small amounts of
lsopropanol (about O. 6 volumes) has been reported tit separate high molecular
D N A from polysaccharides (Marmur, 1961) The sodium acetate also yields
,l tight fibrous precipitate th,it is easily washed and dried The DNA will
dissolve readily if allowed tit rehydrate ,it 4 ~ for one hour fi~llowed by hght
vortexlng

Possible Modifications
The fifllowmg modifications can be apphed to the mlniprep procedure if
problems with nucleases or contaminants that prevent restriction digest of
the D N A are encountered This procedure is also preferred for "difficult"
species such as soybean