0219

racio n o Plant DNA t m , by Dellaov ta Metod

Reagenbs Rauived
-EB,CCtractidn Buey 1)
Pe ite

.S molav EDTA PH8.0

M Tri pH8.0
4M Nac
dH0
BMe (mCoptoetans)

EB2CEtrachion
8 uten 2)
Pew utar-

0. EDTA PH 8:0
M
MTrit pH8.D

dH0

3207. SOS Cdo not awtvclave )- prepane ih hood!

Peo l00ml

SDS

dH0
9M.KoAc_CpotoshUm acotote)
Pe 00ml
KOAC
S) 3M Na0 AC Soclivy
mtcetae)
p e l90 m
Na0 Ac

CtopTbpamot
rotocol 65°c
w a t bath
t Aer at
Prp Make 3Uu

*Deq lg ttistu Cuyoung lual da baby ead PouleRA)

aack onxD in liauid Ny and gsind to poto
i amoYtaN and pestle .JinL can be Stoved in
80 etha bekbn dov atev ninding i aadad
Jtekue 3hould mot thaud boloo E, it added.

*Jotae the tine oodtur into a 0ml Dak Ridge

wbe and add ISmt
8S1
*AdA m
2o% 80so eath tube
o
Maka Sun ibs Aur scuwwed bn tightiy. Miv
thwoughy vigooy haking , and then intubate
tubes in a s°c ater bat or tominuta.
Add Sm SM KOAL
*Mi toyougy by Vgo7out dnaking,thu incubate
tube ar d'c Con ica)
){od 20 min

*Spin Ubes at 13K pm 0Y 20mun in a Se vegak

centuiuge with the &A 60079to» -

(-2goooX ).
Pou Supenatan throug44
MiYacloth(chaseloth)
int 3bmL bak. Ridlge tube tontaininy
l0s mM
iSopopanol
Gnvert 20 imet Ao mix wel and incubate at 20'C oY 30min.
*Spin tubes aE 12RYpm Y ISmin in a SoYval
Cennwge with e SA - iboYotoy (-20000 X)

yenty pouY 4upernatant and ightby dry

DNA peUots y investinq tha.
tub dn ppey
towtes f0Y I0 min

*Rediotve each TDNA peuot ith .7m eb

May med to lut sit overnigw at c ihaving
trolube dilsotving. ansker tnto a
m.endo
tube
*Spin the eppencov tube in a micncontriuge
Y1bi
07 1bminm ar mAN
ar mAy &peod to Temove n&oluble debvis-
*J3antjeY the Super natant tb a ne Eppendo tube
ahd add 1Su 3M NADA C and Sb0ul
isoproponol.
*Mix weu
by inteting 20 time and pellut the DN
30 bo sec in a micokug
Walw pellat with SbDm 801. EtDH dry thourgh ,

and edit50lve in 10 6 L
TE, ED 0 H,0.
Roult
Plant genomic DNA Loas extvacted vom cauufloue
by $ing Dellaota me thool

Ja
 AMTNE MeTHOD
ESTMATOUN OF DA Y DiPHENIYL

do estimat the

Sommpla
Prtnciplu
whEDNA is eared with diphonamne unde
a cid COndit ionl,,A blu Comound i toTmd oith
a h aboyptioh maimwm at Sa Smm. Jhe neacrio
s
i v e by 2-Deoxypentogeg in gneia ang
hor
Specilit 4 DNA (Aderine and yas Uomine ).
aud goution the ehain stiaght rtm foT or a
cdey pentofe sComver ted ti the highly NCactive

-hydroKy levalini c acid whi ch neatcre oith diphny lam

-n to
give a blu Compler.

Matokiols Reaived
DNA Shanda yd &8olwtion'
Sm s DNA
weigmed atturafely
as and make
upto
aSm with TmM Sdium Hychoxidl.

Diphenylamine eaaemt
in 48.7mt
Ditolve . pYe dipheyl amune
gucta Qtwric atid. dd Souoly :Snml o (oncenbYated
glactal
Rulphuic atid Str weu ano 8tore. {0Utuva ute.
Smo
Mateials
Botling ater
bah, petYophotometey, water bath
Test tubes, Jtandavd
MOasuving otk, euvetta
mitvO pt pette

roce duve
¥Pipette Out ilteventr aliapots Dstandavd
DNA SOtutiow in te
Yage d 0Im,0.am
6.Smt with 0. t0 ml wtevve
MaK Conten Oea tobe to Im usUg diltild
water

JD e aliqjuot add Sm of diphanylamine veagent

*Cloe the testr uba t h aluminiu oil and

kap tt fivm by Tublber ba nde

*Hea bn a boiüng watev barth toY 16min, Coot
and Yead th DD value a Snm
RAao u +ea and gtand ayd agai nst watey

olank

0bsey vation-
lue oto ud uY malion is obse) ved

DNA pyent in
he gven Sample
JK anm
DUn b
 Votuw Shuuasi.D
Votur Coneop DNA
Votunme in wovki diphtny
ho.Standard watev Sampl
io u in Yagenb
Solut
ml) ml)
Cmt)
6.90 0 S LOmd0.0

0.80 Om0.0y
2 0.20
10min 0-06
9

t0 0.04
40 4 D.60 80

S O.So, 0.So S 10ma

|0 02
Utnen Bmpe

,
 Scala
X-ocl RUnitRO

O.01

O0f

OiOT

O.D3

O02

20
ftoo

bACOm4anitvation (qat2
131 2l19 Etvathio oAninmol enomic DnA
Pupovatip tdlotion
HomDgeniakion lbu{ler
Na Cl- 1 49

Jris uller-b:1 Aistolue al chemi cals i

aistiued uwatev
EoTA o-01g 00 ml
udY
21 SLS:. 2 SLS in 00ml aistiuad
CSocllu m Loveal Sutphate )

1 9 NaC in 2Smld cReti tlod toater

Ko cold isthopanol

DCedure
homogenizadon
liMey t l0 m l s
DOmg O
OOng O
mOvtas and pestle >add 4DuL G SLS
bu 8oLuon

Gnevbate 65°¢ In

untey
bath 0v n

Ada
300 ul 0
NA CL

Centriuge 19,007pm

0Y D min

Jake Supeinaant
and add eaal
amount d
tLoproponat.
wiu get
DNA
XN RRLul Animal qenomic DA is extyacked nom
nem
ac at liven
 Date loz/19

tutrattiom dateiajeromit DNA

TE -Buktv eDTA in
:
0.1t g t i
Hd f o. 39
bOml disiued
Ho
2 l SLS in lo ml aitt H,
3 phenol: chloro gm
So

44. Sodium acot ate 4.g in lo mi dkUeA

uwod

Procedwu
Sm tk5 bacteruial Cult w .
m
Centyiluge 2màn tor 60o p
Ditcord Supenant

Add 0 o ul a e -bugten

Dixd ve the pouleeby 7lpeated pipeting

-Add 100uts sLS euaaln

37c in
ntubate r ktv at
water kam

add Im o Phonol chlonkoTm

ncuba miNotA
 Contrt Cooopm or min utes

Jako upper layey ond add

too L ols Sodt Cot ouke
ols um

Ado 2m O 180
popamot
it PTopenly DNA 9 t

P1Rupta.
ReLuut
Jhut
Chwrmogornal DAJA is t a ctd
O
acteria uutu

J
 e 1 //19

DnM
etvarion o acteial plmid

fparaion_d Reagenti
Lyai buyt
yweose-0.1 g dinolve. tn 1b0ml
di'stiuod

D T4 0.3gm wate

Jvis o.4 gm

Alkali ne lysis Jouubon

stolves
it
NaOH- 8g Lobm
stiUnd H,0

SLS gm d
l00mC
RSqm di isolve in
3 8oLU acokoute

ocedwe
Ctenial Cultue
Jake Sm
Smi'
Cont
Centrtuge &ooo pm foy

and
DiS Cad soper natant
n lm
the pUuk
euspend
ysi but

7 alkala na yis
Acld 2m
So u u f o w

Sodiumu
Adld 1ml
ite Co d
acetate ouhiom
 it proper y
i

lOmin

AVe on
a
oT

C e n t u a toovo p m

and add
natant
Jake Super
1Soptoponaf

ooubla
he Vo
Lume 0

bath
at
in wnt e r
Mubate

3 fc 3 0 min

gets Pe ipita
DnA
PlAmid

Rosut
Pamid DNA
iS extatted
Jhus

Bactenial Cultuwne
e mo ote60310

unge DA
t r a ton

Pepnaion o Rage ntt

t.EtraCHd But
Jis Hct 1
OTA 018 disiolve in

Don distiwd uwaton

NaCl 0g
SLS 0

tDml! (0 ml
Phenol Chlorotonm
:
2
2.

Take ungal Cultw

Sooovpm or 8min
CLutniluge

Cotutt thepelet and tran

tuhe and
it to amL eppendo
add extracion butr
add (as eauired)
min-Soo ul t may Rmu

at 700m
ymir tt dnd incu bate
temponatun or 30 minutes
in

wator bah

Contritug (0000Pm ou
minutes
 &opnoqnt
in anothe
Onuonthe
tu be ond add eauat
eppendno
OmDUnF chlbraoYm phan ol owhu

My t pealy anol kaep ir

PTO

Cot ot

2win

Joka &Upernaut and add

nal amount o opTpanol

30 minotes at
Koop it 07

PNA oL et Ppt.

Res
DwA it extracted
hos the fungal
ung Cultur.(peg ius nigev)
 on bacterial &uvival
0.v. radioion
o
-

ec
Pro cohuwu
and inno tulate with
noth
e p a r nutvient
Purepane
Lnoculate or 2 Ys
E Cou tultwre

Phepanu Nutient A gr plates

aluL
Cultrw on
&Evika theE.co
plates tin Sam patter
Plare
t on,
tortrol pate
(outep
epoe oL plates
3,(a0 min)
4Y plate , (10 min), plakae
plat y (30mn) , plote uomin), So min
under U . v

tima 'mmedi ately

Abtn the appsop iate
aluminum tot l
Cbver the plantes oith
and ksp in dank toa bvernig ue

Counr the Coloni Ond conpae tha

th
baeteua agaiyst the
mo p hology
COvdro on

2,3 ghowe
tveated plot (plate no na.
sU-V.
-

plake
nUmbon Ohene as
Coni witn
depeane cblony
ivereae i number o
&howed tte C o n tr o l bne
wiphuogy then
itLd
Cplake c).