Restho- Modip'catten Syekeuns

Ecoli c
Badeiopleage d
Co00
Jo Same stoe

2ttuin
0nelud endo nuleaap
hat selettinelly
)

Bateva Hal
boduce RE alo By DNA e-(Meteylaho
pao duce natuylae
ons etuyle
bNA o tiatitusill add
eet be cleamed by to one oto
bases

TypeT Restihn Engues

RMeestrichu
t oRI
eHdduLewe)
EcopLts 24-26 3'to
EcoB

" 2 Biutanal Someeohat

subwila, ehsymeueit ulaeto
Aestsit batti endomucleage
aud
ud meteyle
autiatyy
bbyand modicaiea Two diet
Seaeiees usich Stutwwie
eubuity ae
ae palti'udiru
pallL ndcne
eCovhais3 dilleyet subuniy
stoo
Reqwes
ReaineE ATP Mglt
" deosylmetu'he.e ceaoes at ornas
estnctin
Tuo rdentical subuik tes
 5-4 4A

deaveye paiut Cohayf ve eud on owehau r

shaggered euds
symutet

col (Spees)
RY1b( stra'
ERheuclia (yeuus)

The EcoR I

cleaue e iutenucleahde baud blw

Eo RI speifaltyattuhedto seabou sug t
the plhoshhate
mucieaide ad
It ceetes staggered ud
uecto
teu oe cau nae
aguet hae blut eed
efiueuy tigai
DNA
end b l t but
does not give ligk
Duee
coue t ' s
Eco RI te to eud oy te
So to Cataiw ug
a Swal de Dn
Digete DNA Jahet stageed eud that s
bleut it
eudvetor ueates
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tla
ceupatible tH
Tu's small
staetebeg t
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 euds sidey wt
lele' adapte
aud eud blut the to
tblunt eigateDNA toadaptea deielep o
Adaplers-
Te end va
cohe wth ineeu ale These
speeiidby ueale
wted uigated
uic Veetor
iu aud Iuereyt heuo
ASThes
culer Hexa
cuts itiwes I
cuti
Heat stey Seetneh
Bau
I H NdeI
cud Blunt
TA GTATAC
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AC
4TAT
TAT4 AC
phosphale
aud s' need ’10e
 Tussday
3o"C KJ/uualp ygkophale
ATP

poy

phaplae

igase.
paovde coor he adenylahin peoceys
Sypues
wewch peovde ad aug g
has glutha ade 2tdue
eLkayotes NADp
bactesia-A Te
Teuinal deoryusleotty Fand
- eyase (TDT)

oeneutcloning oas
pelytTails
e Haioaupwication uig pinete
PcR

2Lmbi katau- (Aateoa taung)

Aost (L-R 2e coubrnatrodee
pchauye eIean
ud exehaye
..
 TA Clau'g weteod.
3)

Tewplate PeR oaubi'e

iuddpeudeuck
TOT

T not d h e d

podue TA
veetor
Tueye et hane
Jos T to

,only
don add a y ote mutlest de

Tueu add Td T
ot ru
But TaT doe
hat ou oautoadd ony
pefou

a conto led gM.

XuI

cAAAA AAAAAA mAh

vechor te

chauyesDUA
Toografly
TOPD Comwes u al TA veetor
eusgua called -topoo uaLose
haue nidkig udseig
Vacivia to po isoweewe. paope
No loss orA0moUal
eccTt
I2jut a nick

ToPo1soway e.

eud
tee

wtk itsell T heee ecda to be

Clouing veehers DNA
to thehost cel
Vectors

Casngvetost dilleent ivlems

w
Tey do clig

copica
exta-chomo somMal DNA.
Arst plasmaid to b'e dieovered oas pBR322

PBR922
plaswd

3iudepeudcat nabuasey

Tsehnlchvaa heu jwleuest lod

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pUCSvedoyas (coo-8eo)
pecell

kbpe pat
 claage

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13

Us ed for naking
eiska DNA
aecephibili ss closed
tus
veysall dse of ineted
inyet ea be
a kb
has speialised sequeue
soit cau notmantaiy wich b wd peoteis
J nveled 4dptraked
ee NA

'ngeted ito pUc

pEMBL8

gateiato

deleted

into cell wid

phage ivnyodedEcoRJ Retschw
Cauta

DNA wt be neytieled
plhago
some wwl becme
 Cec cile (en hasi e
ke)

kept o7
at DNM noleute

Leadi to tHe developud Cosuips

bawt?

oscle

-otls veto douelu ped

COs
nd

Cos

B C
Thu

fnmstrs
Aasad phepo hand

paoduo

’bateyal Ecal
pao banohe
5eukanyott

chousd be wgl y
cho ld nol be
T
4ftshould vetr/Aepoytee

t chould hae
eites)
seaueuee.
eukonyoheT chauld hase onal
gbs
 Bacual expesian syheu
Lae Pa otor
Tap omotr

SV 40 ud otteast

The eoo&st nd easie

P.ptons

Bacudo viaeukyohe expseoayo

iwed cell erpeCmo s

Cell ee erprasrlen sylew

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De e hot oteu 14
eulcanohe paleaohe
basod outt odut
De au

beulo yeot Syale

Net all pTM e posable in cae
Sysiea
So, we chooce wawwal
 codrn a

eule
yietd s not

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cali exLacpemou
pmotor

peBmote espra
Veetoy
pET Ta
Tphex
LpPBAD
86 a r b n a e a0mohor

tura mRNA syatys

we bateual sylew
expuespET:. aud

Aaue TTa peomotor (TA palyerase)

Ta palyueae
ream
eseut upst
lac UVs a

Lae poeotoy
mo

Indu ce expem
pTu

0e we batew'al po notur

Po ynee aue pAeut

Pionotuy
 Lysosge
plaud.
To avod
en1tos
Laaky expe tontain chloru awph'to
aubbioh'e meKe

RNA cbdig
to averd codoy
dy biased
swi table s
aplysacodar
exproynau Poreta (0E 30

RoreH an
To s ale
Modtied

heuttia maha Cataetoo e

Tempead e peoblew ou'aled wid otius

Come pR,04e donot Sustana 24°c

poblem

bateva
 meit populb ox peinu sygteu oS l0220 24

Brcelon
lei.
Lspeteik expaan ds

Me hot man
siuulae to bacta en pesiar cye
leu.
yoagt ilh plo mud
traieut ex pre
pcMUYsv40)
uy ed.
V|aal pomo tor ae
eoteayyto get
inatinge fusectus
|dua!
yeasytomavipulate.
geuoue 3 manplated by. ufe.
tutal
mauolaye
fhediu

polyhudin geu podut

oonstrea oy tap
Benoned d ReuovAed.
- hioto’
peo

lou'ng-LR Reeombiuatas
huetlod

ueahan caette.
We end poude
paudbohmauipwaton nathod.
dousr plasmidl
This pacos snde tte tube
trauosh ed ONA
into hast :
vnal DNA iaseyped
The modihied
Commaatceesfcellli
ueo
Taausteai
e neod
to acivale utako waehi t
e an alo be fford

dop by dinp
Thse cel have geneah'eu Hme ot 2hYn cubahu tie)
No a
aye thes e aue alo added Bqal Belue easa

Visus ycdavd eomes out iu meidia. (ar yhu ).

se eesh cel aud stat

Lo,tat cell do not but

MOJ- hus mauy ins pecell,
Ju the vial ttre

Plaque Assat
1cells
beyone
(aad ne

wi e
 4ale tha superad (herau e

T cal culate TiAretu/

bcowt the mora p

Tus
noculu nee ded to add MoI Xtotal ce

Lno ulwm needed

<teget yifauted y

bouday loecues dale diauke 4 2-3

tuces tha
ease
Hee