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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
A Plant D N A Mmtprepatutton. ~,rston 1I 21

1 Tissue ,s frozen m Iiqmd mtrogen and freeze drmd. The lyoph,hzed


t~ssue ~s ground to a fine powder w~th a mortar and pestle.
2. Follow steps 2 through -7 of protocol I.
~, Add 50 I.tl a,M NaOAc and 1(10 I.tl 1~,~ CTAB ICetyl tr,methylammun-
,um brom,de), which w,ll prec,pltate nucle,c acids Pellet the CTAB precip-
itate for :,() sec m the m,crofuge and wash the pellet with 70~'/~ ethanol
~. Rcd,ssolve the pellet ,n .tllll p-I TE Ethanol prcclp,tate the DNA with
50 ILl 2,M NaOAc and 1 ml ethanol
The C T A B - D N A precq~ttate may not entirely red,ssolve but cont,nue w,th
the ethanol prcctp,tatmn
5 Repeat step t The pellet should entirely red,ssolve and the second
ethanol prectpltatlon should remove the res,dual CTAB leawng the D N A m
the sod,urn form
6 Finally red,ssolve the dr}' DNA pellet m 10 mM Tr,s, l m M EDTA
pH8 per gm starting material.
Mmlpreps can be stored fi)r several months w,thout ev,dence of degrada-
tmn and can be cut w~th a varmty of restructure enzymes and hgated w~thout
(urther purlficatmn. We find that lO.O I.tl of mmlprep DNA is suffioent fi)r
a s,nglc 8 m m lane in an agarose gel which is to be used fi)r filter hybr,dlza-
tlon w,th single-copy probes. Heat-treated RNAase must be added to the
rcstr,ctmn reactmn t~ d~gest contaminating RNA m each prep. Hence, a
t}'p,cal reactmn would conta,n the following

bhnlprep DNA 10 01o.l


10X R c s t n c t . m Buffer 3 0p-I
I).5 mg/ml RNAase 2.0p.l
Eco RI 8.0 umts
d H 2 0 to 30 p.l

Digestion is usually complete after 3 hours at :,7 ~ Occasionally, mlnlpreps


are d,fificult to digest w,th certain enzymes. Th,s problem can be overcome
by adding 5 0 gl of 0 1 M spermldme to the entire mlmprep before dlgestmn
Isee Focus 4(3) 12, 19821

References
Davis, R W , M Thomas, J Cameron, T P St John, S Scherer, and R A Padgett
Rapid DNA isolation f-or enzymatic and hybridization analysis Methods m Enzy-
mology 65 q0-i-.t I 1
Marmur, J (19611 A procedure for the isolation of deoxyribonucleic acid from mi-
cro-organisms J. Mol Btol 3 208-218

Recetved Auy,ust 12. I983

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