PRACTICAL
TECHNIQUES
on YOTISANDAMOA3 faNTs t3070wg
DNA
isolation
N9007ZIa
Celllysis 9200 ON01ASTANGANAC
biological method
nse to disrupt cell membrane
enzymes onod aibiaooNla
corininse
-
plants
bacteria lysozyme
980011NX033
enkaryotic cells sappanin
A12BSONN
9TANG2ON
(ITE
physical method
pressuteosmotic
acells are placedin ahypotonicsolution 01019
freeze thaw: thawing (unfreezing) repearedcycies of freezing and
ruptures cell membranes: thronghice crystal formation
mechanical method
ceus
andtoughsampies-nginaot
-
arinding disrnpt plant
nardceuwall presentil
homogeniser shearing:
forcedthronghare
ceil ortissnesuspensions
anarrowspac
DNA
purification N2.: A 1.9 *E
è
Sed ystesta 1ac 2 00 A TO 1g
Chioroformextractron Phenol A1 1A009
toxic longer nsednot
highly
:
INNioNOINBsI gield 2640
mixture
phenol: chioroform
as
highclassifiedgoidstandard
Insedcellaremixed
dneto Javh
by of equal volume
0
HO•E
La remove
protein T
remove
¿
speedcentrifngation1ow celldebris
DNA
concentration
asphenol:Chloroform
distinttphases
by precpitation
notmixwithwater
does 2
nsedto 0.2MNall/ Sodinm can
Acetate of ethanoland2
bevolumes
saltandpercipitateDNA fromsngar
M
NA
layer: DNA
agneons
D92N3Mtsr3 9d
Interface: precipitated protein
phenol : chioroform
190
Kts
Commercial
column
contains of high ton.that bindsDNA
silica membrane in thepresence
-
salt 0f 03 M104: 1D A1T 190MN
impurities arewashed
awan D9EN (o7t017a3y 5/3
10w to rélease
saitbufferis used theDNA 21190g
(elation) 911Sd
101031 *2 20112810M09Imnoa3p
measurement
of DNApurity 3maas
el 3o
ONST3ANSMONSA
RESMIAR7A UAMZ:
1.8 2.0 A26D
/A2go of puveDNA
450nglul )indicatesgoodyield high ( 50pq/ml
7 more
sampieneeded if yield top low
con. or 1/03L3
,restrittionendonucieasesT
natnrailnin bauterja 0cCUr
protett agaihst viral DNA
infeition
nitrogenous
base 21 AMg
group
Phosphate
.boar3c:ADioololo
98N man
vsYd lied tonielbo1esmaits
000 gly dosidic
bond
92INI9O eAnLg
9NIN.302NIL 0in3tasd
deoxyribose
2190
BANACOAC DIONNAINO
nucieoside
triphosphate
bontam TAJiONNG
atvecoanition
CutDNA sitesl specific1910
basesegnence)INadayg
091 8/9 0OY WONPPSNG9TE
retognise 4-gbp sequencesnissant
sites
recognition hove
symmetry: micl
palindro
nsuaily EDO0On1SM
ends0NA
staagered fashion:Sticky C ON12I 1A0Q
Ecki.HindlllPstl.BamMirestriction
enzyme
192ih30gouGN oninsda
S0A0a NDNANOINEOSOTD
9Y SN20908N210 1133
GAATTC 3'
3' CTTAAG
5'
5'
cleavage mechanismm0lTnJT A.40
backbone
phosphodiester cleave
via
E coRI 000in thepresence
of M92t
1S000Io V.E.xot
directfashion: blnntendsSISIN
NNidOSAO gronp
phosphate hy drolyses
Prult.Hpar, Haell:lonsdga NIOM ANDSNO SYAMIS3
19 V0N30 3' 0H 5' phosphate
5'
3'
G5'3
CAGICT
G T CGAC 9V0m31NoitdpNata6 bgagz
r3te to x:m mplds togda do esaNNgJonitaib
hoderistaoisg AA0
cloning IND S1ATJA
MNTA
62 Icoen
pnis.Ojoria 63MNION
AMO
storigxio 014 DA0NNG.M
KpnI
mnitiple clonina
Síte
amp AAN:1906 Saci smuitipie restriutionsite
907197 miSI 092R$10IA5G
enzume
defermine
nsed
number
ori
Adenoviruslbe: EwRlHindlll 0
ELORI
more
genome
smau
4x104
on
10NcT8
depends of fragments formed
restrictionenzymes
P1na
nsed
Sites.Cutat speuificreqion
recognize
Size of
hasfewfragments
genome
: 3, X104
bpboppHuman bpE.colii:
gof
-
ENTATNOS
NONAOO
fsn 10
NATDD9NzawS8230I4NT
IS betterasit cutat lessbol fraomentseN
al iotiNo
AM4C70 109M9IN2SD1TN0
076A A
, thenumber sitescan
of restriction beestimated
GAT C( enzymeSan3Al) 459 5
bases:
4 base
recoanize seqnence | [n 1024
4 -base
Sticky end acutsevern
1024bp
CF ,
fragments prodncedif
enzym
T
®
T
Jy
G
-ove
C
T
recognition segnence
4 bases
9COTSON0GE
fregnency
•
4* Cutsin every256bp
91ANN:JA
SVODW
genomeused
30.12kb 30720 =
to diqest
bpf
31Fragments g 30+l
30.72kbi
807204 1024: 30 cuts
:0070
1T6PP'
e
=
2A N E
10H HG 01
bases 46 : 101KE
in 4096
bp 0GA2N
017050ing .!02
§ bases 46 l 1n65526 9.AM
bp ON M9:AW
0321h
0003637
humanlencocnte 00 30 asotivo n3swisdNo13szAudA3uemA
nucleicacid Stainin:ael can
be seen nnder Vvlight
bromophenol blue tracking dye for visualization
yomMaNSaNSye
cut with wholecen
DNA
Boul or ELoRI
50/150
mlarge genome manyfragment
runqs asmear
DnaN:
P DO MORO can
beidentifred
by Sonthern
blotting
1172 membrane
710092G7
probe
segnence
forspeuificANSE DNA
+00
Aransfer
bacterial virns DNANUS 13mT7 RY000301
cut with Hindill /oco
sman génomeldess fragment
0S1402119072r9000A7I
10SI0
gofDNA
separatin bygei ciectrophoresis
fragments
agarose
polymerized
2503 + 3
18porous
51010
which
270078
auows
movement
108 TH1
of DNASOINEXOM
180I7BN301
smalermoleculesmove
easilu 739 ANCIINS
NOTATEN10
moletuies
Compact move
AINOA easily than lonmer moleculest Anq
9livsta A1riN d bar
linear(ds cnt) 2 102 Oj49sDoio3b tapiml ebnk BME
differentreco@nitionsites
292A0
concatamer 4
DNA
circular joining muitiple.
copiesofSJNSNI
Samè pNAsequence
oli
19MNT 6NM SATESPND
opencircie (nicked) 3
retaxed
less,
piasmid DNA
strand onein
compalt
circle1
Snpercoiled
a
with break(nick)
B NedA
DASANA
Jo0d
6 NATnlo 03mDc
iauml Bk
F1R12
as
•
migratetwisted
faster tightly 00CO
compautmore
, Eovervicw
of PcR 2bt12 008729090 003 %73dmNo
98
-
reagent 84290 MAENAASMNAAL TAD
template sanINAsl2
82Ad 35i0033X
:Specific
targetDNA or
primernl
GDNA tobeamplified
segnence NAN
Dn9Na01232Nd 8s
ras 1
-
nucieotideT.d& 138N A A
thermostableDNA pOL 12T.08 3
TAO,DNApol -(.72°4 )stos 3° potymerase 3
DNA
PFU pol ( 75°c) 1108 activity T T
has3'- 51proofreading autivity
accurate more LUDNSNNSY 191030.A9203Ih 00302
buffer 8 3 24 NMSv3!21N 2said4
Tris HCIpH8.0
maintain
con.
binding primeraffetmart
AEL
optimalpHforSunthesis 29800
water
deionised upto final volume
make 23800 3
PCRreaction
primers amplify DNAseitionbetween the two 9TNUCONSINAIENN
039ION.:
PCR: productInclnde biJa
primer sequenceinte
NOTANINA Ton3:VoGNsNld
reverse
primer 30
outin BUC
Carried LS3
PCR
( sense) 3' 5° 1003-thinSwauedPeR
tubes
matchat3'end to maintain
temperatureJ7:0
optimally AN1 change
edinrole NYSNTNo2 20877I1NSNI NND
3' 2 971.01
900Y _0313902104 t. denaturation
Foaema (q4°)
(anti sense
)
forwardprimer
9JM9 N0.92
anncalina
to separate
(55°c ) 3tand2.
strand
to
annealprimers COo1
(72°Cn) mesus3.extension
SizeamplifiedfragmentthatiS of
O9IS19 0 2TNSMNRT AMg Ao NI1ANA09Z
max cncie
insufficient
:A3.0%0
reagentleft starts
to
lDq amplification phasepg
pratean so
ofter Cycles
L9NINI
GG
*
-
Irmitationof conventional
PCR NienS gvont SUNDSSONI
known DNA
targetseqmence
sterile must be nitra
isrequired
forprimer
desighm
RNA in
primer design
hands
might PNA
degrade
1/
for
0
primer
0391
9N9139920
baseslongsunthetic
lenath minimal
18-24
oliqonncleotide BLAST
.3g
nsed to 1S
specific nottooshort: NSM0TA.M03 design primer
lonatoo slownybridizati0nol AveislNnla
preventprimerdimer
formationul seifannealing
Seif complimentary
pairtheannealing
temp
segnences
mnstbe similarforeach
no
(hairpin)tona
primerIn
"Gccontent
40-607. if possible 00921r0 313173
m9g0
startAnd endwithGlpairs
bond
hydrogen ) Glbond>
is
Strongerbindingprimer
D48s10
stronger
('3
0 NtiwAwgbimanLg
fJogmod
90bhi
0n0773
3asl bsxalsy
common asmostto avDidT
Mismatch
9laxiobal1o313gN2l
137204 p3721wtuptrart an
6000
* Agm03 9Yohu
TECHNIQUES
on YOTISANDAMOA3 faNTs t3070wg
DNA
isolation
N9007ZIa
Celllysis 9200 ON01ASTANGANAC
biological method
nse to disrupt cell membrane
enzymes onod aibiaooNla
corininse
-
plants
bacteria lysozyme
980011NX033
enkaryotic cells sappanin
A12BSONN
9TANG2ON
(ITE
physical method
pressuteosmotic
acells are placedin ahypotonicsolution 01019
freeze thaw: thawing (unfreezing) repearedcycies of freezing and
ruptures cell membranes: thronghice crystal formation
mechanical method
ceus
andtoughsampies-nginaot
-
arinding disrnpt plant
nardceuwall presentil
homogeniser shearing:
forcedthronghare
ceil ortissnesuspensions
anarrowspac
DNA
purification N2.: A 1.9 *E
è
Sed ystesta 1ac 2 00 A TO 1g
Chioroformextractron Phenol A1 1A009
toxic longer nsednot
highly
:
INNioNOINBsI gield 2640
mixture
phenol: chioroform
as
highclassifiedgoidstandard
Insedcellaremixed
dneto Javh
by of equal volume
0
HO•E
La remove
protein T
remove
¿
speedcentrifngation1ow celldebris
DNA
concentration
asphenol:Chloroform
distinttphases
by precpitation
notmixwithwater
does 2
nsedto 0.2MNall/ Sodinm can
Acetate of ethanoland2
bevolumes
saltandpercipitateDNA fromsngar
M
NA
layer: DNA
agneons
D92N3Mtsr3 9d
Interface: precipitated protein
phenol : chioroform
190
Kts
Commercial
column
contains of high ton.that bindsDNA
silica membrane in thepresence
-
salt 0f 03 M104: 1D A1T 190MN
impurities arewashed
awan D9EN (o7t017a3y 5/3
10w to rélease
saitbufferis used theDNA 21190g
(elation) 911Sd
101031 *2 20112810M09Imnoa3p
measurement
of DNApurity 3maas
el 3o
ONST3ANSMONSA
RESMIAR7A UAMZ:
1.8 2.0 A26D
/A2go of puveDNA
450nglul )indicatesgoodyield high ( 50pq/ml
7 more
sampieneeded if yield top low
con. or 1/03L3
,restrittionendonucieasesT
natnrailnin bauterja 0cCUr
protett agaihst viral DNA
infeition
nitrogenous
base 21 AMg
group
Phosphate
.boar3c:ADioololo
98N man
vsYd lied tonielbo1esmaits
000 gly dosidic
bond
92INI9O eAnLg
9NIN.302NIL 0in3tasd
deoxyribose
2190
BANACOAC DIONNAINO
nucieoside
triphosphate
bontam TAJiONNG
atvecoanition
CutDNA sitesl specific1910
basesegnence)INadayg
091 8/9 0OY WONPPSNG9TE
retognise 4-gbp sequencesnissant
sites
recognition hove
symmetry: micl
palindro
nsuaily EDO0On1SM
ends0NA
staagered fashion:Sticky C ON12I 1A0Q
Ecki.HindlllPstl.BamMirestriction
enzyme
192ih30gouGN oninsda
S0A0a NDNANOINEOSOTD
9Y SN20908N210 1133
GAATTC 3'
3' CTTAAG
5'
5'
cleavage mechanismm0lTnJT A.40
backbone
phosphodiester cleave
via
E coRI 000in thepresence
of M92t
1S000Io V.E.xot
directfashion: blnntendsSISIN
NNidOSAO gronp
phosphate hy drolyses
Prult.Hpar, Haell:lonsdga NIOM ANDSNO SYAMIS3
19 V0N30 3' 0H 5' phosphate
5'
3'
G5'3
CAGICT
G T CGAC 9V0m31NoitdpNata6 bgagz
r3te to x:m mplds togda do esaNNgJonitaib
hoderistaoisg AA0
cloning IND S1ATJA
MNTA
62 Icoen
pnis.Ojoria 63MNION
AMO
storigxio 014 DA0NNG.M
KpnI
mnitiple clonina
Síte
amp AAN:1906 Saci smuitipie restriutionsite
907197 miSI 092R$10IA5G
enzume
defermine
nsed
number
ori
Adenoviruslbe: EwRlHindlll 0
ELORI
more
genome
smau
4x104
on
10NcT8
depends of fragments formed
restrictionenzymes
P1na
nsed
Sites.Cutat speuificreqion
recognize
Size of
hasfewfragments
genome
: 3, X104
bpboppHuman bpE.colii:
gof
-
ENTATNOS
NONAOO
fsn 10
NATDD9NzawS8230I4NT
IS betterasit cutat lessbol fraomentseN
al iotiNo
AM4C70 109M9IN2SD1TN0
076A A
, thenumber sitescan
of restriction beestimated
GAT C( enzymeSan3Al) 459 5
bases:
4 base
recoanize seqnence | [n 1024
4 -base
Sticky end acutsevern
1024bp
CF ,
fragments prodncedif
enzym
T
®
T
Jy
G
-ove
C
T
recognition segnence
4 bases
9COTSON0GE
fregnency
•
4* Cutsin every256bp
91ANN:JA
SVODW
genomeused
30.12kb 30720 =
to diqest
bpf
31Fragments g 30+l
30.72kbi
807204 1024: 30 cuts
:0070
1T6PP'
e
=
2A N E
10H HG 01
bases 46 : 101KE
in 4096
bp 0GA2N
017050ing .!02
§ bases 46 l 1n65526 9.AM
bp ON M9:AW
0321h
0003637
humanlencocnte 00 30 asotivo n3swisdNo13szAudA3uemA
nucleicacid Stainin:ael can
be seen nnder Vvlight
bromophenol blue tracking dye for visualization
yomMaNSaNSye
cut with wholecen
DNA
Boul or ELoRI
50/150
mlarge genome manyfragment
runqs asmear
DnaN:
P DO MORO can
beidentifred
by Sonthern
blotting
1172 membrane
710092G7
probe
segnence
forspeuificANSE DNA
+00
Aransfer
bacterial virns DNANUS 13mT7 RY000301
cut with Hindill /oco
sman génomeldess fragment
0S1402119072r9000A7I
10SI0
gofDNA
separatin bygei ciectrophoresis
fragments
agarose
polymerized
2503 + 3
18porous
51010
which
270078
auows
movement
108 TH1
of DNASOINEXOM
180I7BN301
smalermoleculesmove
easilu 739 ANCIINS
NOTATEN10
moletuies
Compact move
AINOA easily than lonmer moleculest Anq
9livsta A1riN d bar
linear(ds cnt) 2 102 Oj49sDoio3b tapiml ebnk BME
differentreco@nitionsites
292A0
concatamer 4
DNA
circular joining muitiple.
copiesofSJNSNI
Samè pNAsequence
oli
19MNT 6NM SATESPND
opencircie (nicked) 3
retaxed
less,
piasmid DNA
strand onein
compalt
circle1
Snpercoiled
a
with break(nick)
B NedA
DASANA
Jo0d
6 NATnlo 03mDc
iauml Bk
F1R12
as
•
migratetwisted
faster tightly 00CO
compautmore
, Eovervicw
of PcR 2bt12 008729090 003 %73dmNo
98
-
reagent 84290 MAENAASMNAAL TAD
template sanINAsl2
82Ad 35i0033X
:Specific
targetDNA or
primernl
GDNA tobeamplified
segnence NAN
Dn9Na01232Nd 8s
ras 1
-
nucieotideT.d& 138N A A
thermostableDNA pOL 12T.08 3
TAO,DNApol -(.72°4 )stos 3° potymerase 3
DNA
PFU pol ( 75°c) 1108 activity T T
has3'- 51proofreading autivity
accurate more LUDNSNNSY 191030.A9203Ih 00302
buffer 8 3 24 NMSv3!21N 2said4
Tris HCIpH8.0
maintain
con.
binding primeraffetmart
AEL
optimalpHforSunthesis 29800
water
deionised upto final volume
make 23800 3
PCRreaction
primers amplify DNAseitionbetween the two 9TNUCONSINAIENN
039ION.:
PCR: productInclnde biJa
primer sequenceinte
NOTANINA Ton3:VoGNsNld
reverse
primer 30
outin BUC
Carried LS3
PCR
( sense) 3' 5° 1003-thinSwauedPeR
tubes
matchat3'end to maintain
temperatureJ7:0
optimally AN1 change
edinrole NYSNTNo2 20877I1NSNI NND
3' 2 971.01
900Y _0313902104 t. denaturation
Foaema (q4°)
(anti sense
)
forwardprimer
9JM9 N0.92
anncalina
to separate
(55°c ) 3tand2.
strand
to
annealprimers COo1
(72°Cn) mesus3.extension
SizeamplifiedfragmentthatiS of
O9IS19 0 2TNSMNRT AMg Ao NI1ANA09Z
max cncie
insufficient
:A3.0%0
reagentleft starts
to
lDq amplification phasepg
pratean so
ofter Cycles
L9NINI
GG
*
-
Irmitationof conventional
PCR NienS gvont SUNDSSONI
known DNA
targetseqmence
sterile must be nitra
isrequired
forprimer
desighm
RNA in
primer design
hands
might PNA
degrade
1/
for
0
primer
0391
9N9139920
baseslongsunthetic
lenath minimal
18-24
oliqonncleotide BLAST
.3g
nsed to 1S
specific nottooshort: NSM0TA.M03 design primer
lonatoo slownybridizati0nol AveislNnla
preventprimerdimer
formationul seifannealing
Seif complimentary
pairtheannealing
temp
segnences
mnstbe similarforeach
no
(hairpin)tona
primerIn
"Gccontent
40-607. if possible 00921r0 313173
m9g0
startAnd endwithGlpairs
bond
hydrogen ) Glbond>
is
Strongerbindingprimer
D48s10
stronger
('3
0 NtiwAwgbimanLg
fJogmod
90bhi
0n0773
3asl bsxalsy
common asmostto avDidT
Mismatch
9laxiobal1o313gN2l
137204 p3721wtuptrart an
6000
* Agm03 9Yohu
