PLANT MOLE('ULAR BIOI.OGY REPORTER Volume 1, Number .

t, Fall 198~ EXPERIMENTAL PROTOCOLS

pages 19-21

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 (Dellaporta 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 apphed 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. However, the procedure reported here was selected to be statable for most situations. 19

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Plant ~,lolecuD1r B1,,D4~ Rep,rto" 3

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 insoluble 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 isopropanol 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 microfuge 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 precipitate 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 degradatmn 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,dlzatlon 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 10X R c s t n c t . m Buffer I).5 mg/ml RNAase Eco RI d H 2 0 to 30 p.l 10 01o.l 3 0p-I 2.0p.l 8.0 umts

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 Enzymology 65 q0-i-.t I 1 Marmur, J (19611 A procedure for the isolation of deoxyribonucleic acid from micro-organisms J. Mol Btol 3 208-218

Recetved Auy,ust 12. I983