}Y /h&"'

DNA ISOI-ATION

INTRODUCTION

the
Nucleic acids (DNA AND RNA) are the l'ltal macmmoleculesin aI living cells ifre nle contains
and is iocatedwithin $e cell. The efficiencv and recovery of extractiond€pends
'ti"-*."t1" i"i..-"ti".
i""-1"
.r ru.of* ."t".ia, suitablemedium, ionic condition of tbe €xtaction m€dium, t}pe of the lysing
u""a'"tc. e*t uctio" of DNA is acconplishedbv the rupturing of cell watl and nucl€ar membrane
f;bwed by depmteinzationandprecipitationofthe nucleic acidsusing ethanol'
-"ni

we use two Drotocolsfor DNA isolation. The Qiagen DNeasvKit is quick and easv' but the DNA vield
ma\ be los. and lou trlay experimceProblensqith eg pollsaccharides The CrAB Prorocolis nme
.;;;; il;.';ito',,pui i. e*.,;rrv high.eerore isoiationvou musr weisb voursampre\You will
.""J ti' zO .e a'ied pl;t material for the Qiasen protocol (30-50 Ing for CTAB) If vou are
'p n". n"!ft "ili* to l0 mg material ('iried or
i".iuring to. a.zen)-matenal, llse up to 100 mg For alga€,use up
fiesh). work assterileaspossible.

(n\ DNA bobnon usingthe Qiryen DNeas! Kit:

7-10 daysold culturesuspension(biomassbetwe€n50-60 mg) was usedfor the extractionofDNA

(2 nrl).
CeoFfugesuspension aL14000rymmin for l0 mins
Removedsupemantwith the help ofnicropipette slowly andretainedthe pellet'
Centrifugedagainandcollect supemafltwith the miootips
petter. -eaae'a500 pl butrer(sp€rialbuffer r itb lvso/ymer:lodexed oicel) atrdaddhg !hi"'
be
inlubared at 37"C for 30 mins. To I tu.. wbile in incubation!/as going o& mixtut requiredto
vortexed2 to 3 t1BGs.
. Centrituged again carefirlly (sane spe€d)and r€moved the whole supemantwith the help
micropipette(Entire to be removedcarcftlv).
. Pellelwassuspended in 180Fl A IL bufferUrrovided in theUt). voaerediLaeain'
. Add 20 d o;teinase analvortexeal once again atrd kept for jncubarionovemight at 55'C Thrs
needsto be vortexedawaveveryhalfan hour till possible'
was
. After ovemight treatment with prot€inase K and ATL buffer, treatmenl with RNAase
.equired.
. Add 20 pl RNase(2ome/ml) ftom stock available;mixed thoroughly bv votexirg and imubated
for 2 mins. at room temPerature.
. Vortexedaeain for 15 ;econdsand added200 pl buffer 'At' 10the sample,mixed thorowhlv by
voftexing a;d incobatedat 70'C temperaturefor | 0 mins'
. Added zuu
AOOeC 200 pr pl eLurur (96-1000/0),
ethanol(to-rvu7o/, mixedthoroughly
!D^!u uwlw by vortevjng'
. Transferreal the cofients on the top ofDNeasy mini column in a 2 rDl collectionlube C€Irl fug€d
at 8000rpm for I min. Discardedflow througlt andcollectiontube'
rU""a tl" in a new 2 nl collection ti'be {provided wjth kirll ailded500 Fl.Awl
iutfr ;ded) and centituged for 1 min for 8000 ryl| Discarded flow through and
-ini"pio "otu.tt
"tttnof
collectiontube-
(\tith
. Placealaeain minisDin colunn in a 2 ml collection tube and adddd200 Fl df butrer AWz
u?a"Jl, g"a for 3 mins at 14000 rpm to dry the membran€ Djscarded flow^$rough
"*r*"f "*i.inrf,i" can be firther c€naitugedfor I min at 14000rpm to get rd of ethanol
tube.
".J
tully.""i""tl*
Eluiion ofDNA fton column: Elution buff€r (AE): The r€quiredamountof AE buffer sbod+.ba
rr'tuer
keDtat 70'C for 5 mins Placed 100 d of walm AE buffer oo the top of tninispm colunn'
*Jr" tJ or r.s tof microcentrituge.The tubeswere kept at 70.c for 5 mins Spimed for 1
"f"*
min. at 8000 rpm and savedthe flow tkough i! bottom
Asain addedi00 ul ofwam AE butrer or top ofthe column,kePtin incubatorfor 5 mins at 70"C'
ce;trifuged at 80ti0 rpm for 2 mins
 Djscardedthe minispin column and savedDNA in bottomtube.
Comoonentsof soecialbuffer usedwith lysozlmes:

Stock availabl€ Voluoretak€nup {Tdtal volune (5 nl)}

a) lM Tris HCl, PH-8 100N

b) 0.s M EDTA, pH-8 50Fl

c) 1.5M sucrose 1.5ml

d) Lysozlme 50mL

,...1r-')
(b) DNA isohnonusins (Ce,rltimethrltutmonitmbnnue) nahoa |AJF
'TAB
(Give hr Rogen and Bendbh' 1988)

Furpai isolateslTrichodemo) w€re mainiainedon broth media at 25'C and mvcelia for DNA
at
extriction r.reregmwn in liquid culturesfor 2 davs at mom tempemturcwith rotatory shaking
120 ryn. wlre; bactdia at standardmedia for 2-3 days at 3? "C witb rotatory shaki4 at 120

Mycelia/ bactoial susp€nsionwere harv€stedby filteration tbrough a piece of filter paper and
\rashedwitb distilledwater
t roolai ti"",re o. bacteFialtissue (50-100 mg) were gromd h 400 Il of CTAB, whiti .
maiitainei a roralvofumeof?00pl
-y"etlar , ,.-a,r,r.. trcrty'
, ^r )
y'61'n4 -j1
Ilerubeswererhenincubaledalb5"Cbwaterba$for15mins fl"r' 0' " 241 |
Affer a briefcooling700Fl {equal volume) ofcbloroform was ^r
added mix well bv genrlvshaking
a +
in oorca rotaloryshakerat roomtempemture [or l5 mini'
Thetubesw€rethenspinat 14,000lpm lbr l0 mins.to getthetop aqueous phas-
Ft'Qhdl':'
without disturbing it" i.tt".fu"" above the chloroform layer, aqueous phase-(600 pl)
Yt ,Lt*o.(,(I-
trdnsfenedto a fresh I .5 ml eppendorftube(the partistnatematterwas avoidedwhile Pipetting)' I
To the aqueousphase900 pl of 100%ethanolwas addedand\lzs thenmix€d gently ! Eo'rfi
aLl '
nre tutes were ctrnea in -20'C d€ePlieezer for 1'/, hts
Spunfor 3 mins. at 1000 rpm and th; supemantwas discarded- :2e : I
and ,{
l;e pelletwaswashedwilh 70p" elhanol andair driedand was resuspended in 50 IITC bumer
ir,."';ncubateAwith RNaseI d (20 Ing/nt at 37"C fm I hr. andwere storedat -20"C for turther

heparudon ofslock sohrlionsusedin the aboveetpefime"l:'

a) !M Tris HCI (pH-s.0): l2l.l g Tris HCI + s00 r[ djsti[€d water and pH adjusitedbv adding
conc.HCI or NaOH pellet to the volume litre of I lit'
b) 0.5M EDTA (pH-80): 186.1g EDrA + 800nL distilledwater

Adjust the pH to 8 with NaoH pellets(iou will needaboutabou120g) or NaOH solution'

the pH is aboutS Bring volume up to
rhe reasonio aalusithe pn is that the EbTA will not djssolveuntil
ii*rur aootf"ii"tiff"a *ater (ddH2o), pu1 in vess€l
or contaircr' and autoclave'

cl CTAB extmctionbutr€r (l lit.)

20 e C IAB {CetrylTrirnelhylAmnoniumBromide)
 860 tr[ steriledoubledistill water
el82 sNaq u-
100mL 1 M Tris HCI
40 mL 0.5 M EDTA

Slir vigorously on a magnetic sti[€r. Store in Room temperaturepdor to useadd2} p"]/21ml

2- Mercaptoethanot.

(d) Ethanol

G) T.E. buffer (1 lifie) (pH.-8.0):l0 nl- I M Tris HCI + 2 tr[ 0.5 M EDTA dissolvein I lirre
of double distilled water.

l-X TdE buffer

Put 48.4 gm oi Tris base, 11.4rnl of glacial acetic acid and 20 ml of0.5 M EDTA inro 1000 mt of
volumeaic flask or gmduatedclinder. Add distilled \a?ter ro make a rotat votme of I litr€. This
fo.{s a lox stocksolution.Dilute the 10X stocksolutionto makea tX solution(100 n of 10X stock
solurionlo malea lX workingsolulioo.

(a) D N A i s ol ari onus i ng p o n r € r , s m e r h o d lg - . + lf *

(Porter ur,dt 198Gr988) -..
'ld' r",^-r_ &&U CX
Furyal isolates (Trichodema sp_)werc mainraircd on pDA at 25.C and mvcelia for DNA
extsacaionw-eregrown in potato dextrosebroth m€dium for rwo dals at roorD [mp€rature with
rctatory shakerar 120 rpm, whereasbasterizPseailonozar sp. were g.own on King_,s-Bmedium
grorn for 2-3 daysat 37"C with rotatory shakershakingat 120rym.
The cultureswere homogenizedusinga glasshomogeniserandusedfor the exhactionofDNA_
Cert ifuge the susp€nsionat 14,000rpm for l0 min_
J
Removesupematantwith the help of miclopip€tte stowly and retain rhe pe et (biomassof pelet
shouldbe in between50-70mg). 1
To the pellet add600 Fl ofsxedle lysis buffer (10 % suclose,100mM EDTA, 50rDMTris HCl, pH
- 8.0)and!onexnicel).
5 Fl of Lysozlne (5 ng /rnl) was addedto the lysis buff€r containingce s and incubatedai 37oC
I
for 30-45min.
. Add 3Fl RNAase(20 mg/ml) andircubate at 37'C for 5 min.
. 60 pl of l0% SDS and 6 Il of Foteinase-K (10 mg,hrl) were then added to eppendorftube
containingthe lysateandincubatedal50"C ovemight.
The lysate was given phenol ghlomfom: isoamyl alcohol :24:1)
11)treatment(at lhis treatment
with the equalvolume dsthe volume aheadyin thl tribe).-
Thenvortex and spin at 14,000rpm for 5 min.
Traisfer the aqueousphase10a ftesh sterile2 ml eppendorftube.
l/10- volume of3 M Sodiumacetate(pH: 5.2) was addedto the supemarantanalmixed properiy.
DNA was Fecipitated with ice-cold ethanol by placing the tube a1 -70.C for 3 bls (if the rub€
allows,add2.5volumeof 100%ethanol).
Thenpelleting is doneby spinningat I 4,000rym for 40 min at room remperaru€.
Thele et waswashedwirh 70% ednnol anddried.
The DNA wasrccofftituted in 50 Ul ofsterile TE butrer (pH = 8.0),
The concetrtrationand quality of DNA \rere cbeckedby Specnophobmerric readinesr 2o0 om
andby running on 0.8% agarosegel.
This DNA wasusedfor PCRwithout furth€r pudfication \ <]\
RNA ISOLATION
g by filtration on
Harvestlog-phas€cells by centrifugationat the desir€dtemp€rarureat 5000 tor
WhatmanI MM paperfor somefilamentous strans.)
(pH 7 8) and
n""""p-a *" *if. qtl"*ly in a small voliime of 50 mM Tris-HCL' 20 mM EDTA
oiDenethesolutiondropwiseinto liquid nitrogeD'
ff;"i ,r'r"tor* t" u tne powder in a bleoder,or with a chiled monar andpestleaddingmore
""rl
liquid nitogen asnecessary.
rnM Tris-HcL 0 ]
,A.iJth" sro:'-a ce[" to tul equalvolune ofpre-warmed(37"C) lvsis butrer [50
i" N"ii"zo z%;/v sDS. 60 mM p-nercaptoethanol l0 ug/ml FoteinaseK (pH 8)l'
lncubalear J7'C",la-inre.
lor l0 mrn
Ifthe solution is viscou at this stage,passit severaltimestbrougba s)fltr9e
lysis
Extract the mixture thrc€ (or more) times: wi$ an equal volume of .buff€Fsaturated
vvi. e cur' cotourless, aqueous upper laver should be obbainedNo
O,r,
"il"J'v"lt"*-i.""
i""Jr J.rra be ui"itri tie aqueous-organic interfaceduring dre final extraction
'lemove ", andadjustto 0.5 M amnonium aceta$'
the aqueous layer
for a f€w
it".l.i"," rr'J nN,t t:" addingi S volts of absoluteethaIlol and leaving at 70"c
minutes,or l0"C for a few hours'
. Recoverhe nucleic acid bv centrifugationat 12,000g Drain the pellet well -
art
. Dissolv€sthe pellet in st€riledisti[€d water or as suitabl€buffer- Stoie -20"C
.nNn"anno*t"j}eeilofcontaminationDNAbytreatnentwithDnasel,peletingthroughcsCL
otprecipitation with 2 M LiCl.
should' ideally' be denaluredbv
To prevent $e forrnation of base-pairedse€ondarysb'uctues' RNA
in gels
deoarurfug sucb as thosecontainingfomuldehvdeln
nwo'-ru,;* p;- t a..ttophoresis.or run in
ii# srep
.ii.'.ii ii,i il iots'ibtero omira denaturing incuningany obviousdisadvanLaPes
"irnout
tem$ of anomalousmigation of aParticulartranscriptduringelectrophorcsis'

2) QUANTIFICATION OF DNA & RNA

frinciple:

(a) DNA electrophoresisin agarosegel:

Honzontal
i;._J"" .r Oie can be icbjeved in potiacrytamideor most prefemble-asarosesel.
tr," ua'*tug'; lower conceftration of agarose can he utilized
#ffi;;;;;;;hut of the
utto'oiog tti" 'hut -*b of last DNA fi-asments and also handline
;il; "epa'ution on fie
egii..J fonn a gel-bv hvdrogenbondinsald
"";:;i"'J;Jile- r-begel. pore si4-depends
*J Ii.*r"r. on lbe ba\is oflheir
i"".".*,r"" .r?g".* The oNA molecule"'are sepan;d by electrophoresis
ofnet charg€on the nolicules The dve ethidi m bmmide intercalates
light lnw
"ize,-"Lp"-LJ
l"'i'"*irr" ir ruA *a one anil fluorescentorange wh€n irradiated.with Uv
tut""-is'r*a"
molecules
ug".o"" g"l with large poie pflnits ftactionarior of high molecular weight
"oo"-tutlon
andvice_versa nm on 0 8%
ili o"-#i'*i"" stock mix lul' 6X loading buffer with 5ul of DNA stock and
"t "r',o at 50-100 v using lxrAE butre!
i;;;;;);;;s€ lel stainedwith ethidiun bromldefor lhr baDd to a standard
the htensity of DNA
DhotosaDhand estimateDNA concentrationby comparing
mark€;n-DNA) cut with Eco.Rl,/HindIII
 Sand size(bp) Concentration (ng/sFl) Concentration (trglpl)

21226 218.8 43.8

5148 53.1 10.6

4973 51.3 10.3

4268 44_O 8.8

3530 36.4 7.3

202',1 20.9 4.2

1904 19.',7 3.9

1584 16.4 3.3

1375 t4.2 2.8

941 9.8 1.9

831 8.6 1.7

564 \.2 o.2

Iimbda DNA cut with EcoRIdirdII

The extra.ted DNA preparationswere quatified by taking absorbanceat 260 nn. The valrc of t
absorttan€eat A,60is equal to 50 Fg/ml for standardDNA. The int€gity of isolated!:enomicDNA was
det€mrinedby 0.8% agarosegel elecirophorcsisagainst I kb mo16€ularweight for I br. at 75 volts. To
checkthe pudty of DNA , the absorbancewas read at 260 nm and 280 nm andthe ratio of A,@ and Aeso
wascalculated which shouldbe about I .8 for oure DNA.
(r^u StriJH, cerf*"<,prh r/"uta'/'t'i
"t,rit +",'4
A*o at a a"t"'Xod't' /:)t a ruu*l l*f**:

;r;'m*; tr #r#;'fuffiffi7
nwoueot4V P:ffT
ltu rt/</r'14
K*;;-/r
auv. T n'wp * 'i L0o-11'*''
^
J) ffi Tilitila $H's'o)
dd'Lt
tt't gn ,tP f J 00 ml al oblhlltd Palzn
Add $ THtu o .9
adatiT I'faon haltct to t L
& PH ^E'*t"t 4
Ueltttr. + Jnil '

, o'5n EbrA(rH=S'oJ d4 , atata

lhr;tgn FDuAf lr,ont g?idbd '
*tu t0 0 w;r Nlon til'tt on NolH 'tlbxb
F+ie PH
fr ttn P't ) fi''ot ttu *trrl nfu
Tiz n"otco 4W ua/uiv
ttv pn a ot'ld 8 ' i-"j
not ditlalua p*t'
a1 A d*-;o ctoil'h cltttua P:@t
(toO*;
sl
J
c7hg eAtudrrilh'4*
crf\B f*'duz -"
I W *
g6 ,}flt* wota- I
Ao 'r'-- 0
-g'tg ./- \
q" Ncca /' )
o'Srt EDTA '-/
h r^t g
l H 'Ivr bH c t
tornl{ fr*:':::
.,, ^---,,er,g11 tt ffiach|
n;,"9! Lo'at/zct&
X' aatd
il,^;
* 2' lflucalh'fl'aet'
4'ffi 6W
Lr,tt !:]T"* 11
\
o'2N t a'5 t ' l € ' b T A .l

;^ lta lr.t % ]Dt^/ ' f.4'^ / )

lp,arotr.