• Design • Construction • Characterization
•D
esig
n
•C
onst
ruct
ion
•C
hara
cter
izat
ion
CC
AAC C
His
DN
AD
NA
mR
NA
mR
NA
(mes
seng
er)
(mes
seng
er)
prot
ein
prot
ein
transcription
translation
AT
GC
TA
CG
•re
plic
on•
copy
num
ber
•in
com
patib
ility
•se
lect
ion
mar
ker
prom
oter
prom
oter
trans
crip
tion
term
inat
or
trans
crip
tion
term
inat
or
sele
ctio
n m
arke
r
your
gen
e of
in
tere
st
orig
in o
f re
plic
atio
n /
repl
icon
Pla
smid
s
mul
ti-cl
onin
g si
te
Exp
ress
ion
Pla
smid
Pla
smid
s
Chr
omos
omal
In
serti
ons
vs.
De
Nov
o D
NA
Syn
thes
is
OO
O PO
-
O
CH
2
O
HO
CH
2B
B
5’ e
nd
3’ e
nd
DN
A h
as d
irect
iona
lity:
5’ G
ATC
3’
is d
iffer
ent t
han
3’ G
ATC
5’
DN
A is
che
mic
ally
syn
thes
ized
3’ t
o 5’
DN
A is
bio
logi
cally
syn
thes
ized
5’ t
o 3’
De
novo
DN
A s
ynth
esis
is c
urre
ntly
lim
ited
to ~
200
ntD
ue to
erro
r rat
es in
syn
thes
is m
ust p
urify
ove
r ~70
nt
For d
e no
vo s
ynth
esis
ove
r 200
nt,
mus
t use
adv
ance
d st
rate
gies
suc
h as
as
sem
bly
5’
5’3’
3’te
mpl
ate
dena
ture
(~94
C)
anne
al (~
45-6
0 C
)
elon
gate
(~72
C)
dena
ture
(~94
C)
anne
al (~
45-6
0 C
)20
-35x
elon
gate
(~72
C)
PC
Rpr
imer
s
PC
R re
quire
s: te
mpl
ate,
prim
ers,
ther
mos
tabl
epo
lym
eras
epo
lym
eras
es v
ary
with
fide
lity,
edi
ting
activ
ity, s
peed
•P
rimer
s sh
ould
hav
e Tm
sbe
twee
n 45
-65
C•
Prim
er s
ets
shou
ld h
ave
sim
ilar T
ms
•P
rimer
seq
uenc
es fl
ank
the
regi
on to
be
ampl
ified
, thi
s m
ay li
mit
prim
er s
eque
nce
spac
e•
Prim
er s
eque
nces
sho
uld
be d
esig
ned
to m
inim
ize
(i)
hom
odim
eriz
atio
n, (i
i) he
tero
dim
eriz
atio
n, (i
ii) h
airp
in
form
atio
n
•P
rimer
des
ign
shou
ld ta
ke in
to a
ccou
nt n
on-s
peci
fic
bind
ing
to o
ther
regi
ons
on th
e D
NA
tem
plat
e (lo
nger
is
not a
lway
s be
tter)P
rimer
Des
ign
(i)(ii
)(ii
i)
brea
k in
to o
verla
ppin
g pr
imer
s
Mod
ifica
tions
to P
CR
1. A
dditi
on o
f new
seq
uenc
es w
ithin
prim
ers
2. P
CR
ass
embl
y
3. O
verla
p ex
tens
ion
PC
R
PC
R
PC
R
inse
rt
vect
or
Inte
rmol
ecul
ar re
actio
n(e
ffect
ive
at h
igh
conc
entra
tions
)
Intra
mol
ecul
ar re
actio
n(e
ffect
ive
at lo
wer
con
cent
ratio
ns)
Liga
tion
reac
tions
1 of
man
y po
ssib
le p
rodu
cts
–lin
ear,
circ
ular
, hom
o an
d he
tero
dim
ers
5’P
5’P
5’P
5’P
3’O
H3’
OH
3’O
H3’
OH
Clo
ning G
A A
T T
CC
T T
A A
G5’
P5’
P3’
OH
3’ O
H
5’ p
rotru
ding
end
s (E
coR
I)
A A
T T
C G5’
P5’
P3’
OH
3’ O
HG C
T T
A A
C T
G C
A G
G A
C G
T C
5’ P
5’ P
3’ O
H3’
OH
3’ p
rotru
ding
end
s (P
stI)
GA
C G
T C
5’ P
5’ P
3’ O
H3’
OH
C T
G C
AG
Clo
ning
Non
-dire
ctio
nal c
loni
ng (s
ingl
e re
stric
tion
site
)
Eco
RI
+E
coR
IE
coR
I
dige
st+
ligat
eor
Eco
RI
Eco
RI
Eco
RI
Eco
RI
Clo
ning
Dire
ctio
nal c
loni
ng (t
wo
non-
com
patib
le re
stric
tion
site
s)
Eco
RI
+S
alI
Eco
RI
dige
st+
ligat
e
Sal
I
Eco
RI
Sal
I
Blu
nt e
nd c
loni
ng…
C T
T A
A
•K
leno
wfra
gmen
t –us
ed to
fill
in 5
’ pro
trudi
ng
ends
•E
xonu
clea
seI –
rem
oves
sin
gle-
stra
nded
nts
in 3
’ ->
5’
•P
hosp
hata
se–
rem
oves
5’ p
hosp
hate
on
a D
NA
stra
nd
A A
T T
C G5’
P5’
P3’
OH
3’ O
HG C
T T
A A
GA
C G
T C
5’ P
5’ P
3’ O
H3’
OH
C T
G C
AG
A A
T T
C G5’
P5’
P3’
OH
3’ O
HG
Tran
sfor
mat
ions
Che
mic
al m
etho
ds
Phy
sica
l met
hods
ice
cold
sal
t sol
utio
ns, h
eat s
hock
; effi
cien
cies
~10
5 -10
6co
loni
es/u
gsc
DN
A
ice
cold
sal
t-fre
e so
lutio
ns, e
lect
rical
cha
rge;
effi
cien
cies
~10
9 -10
10co
l/ug
scD
NA
Scr
eeni
ng
Sal
IE
coR
I
Res
trict
ion
map
ping
Sac
I
1500
bp17
00 b
p
2000
bp
Bam
HI
1200
bp
4000
bp B
amH
I
LE
/S/S
B
Col
ony
PC
R
Seq
uenc
ing
Con
firm
cha
nge
in P
CR
frag
men
t in
clon
ed c
onst
ruct
Con
firm
seq
uenc
e of
clo
ned
fragm
ent
dist
ance
btw
nen
d of
prim
er a
nd s
tart
of
desi
red
sequ
ence
~50
-70
nt
Alte
ring
DN
A S
eque
nces
Rat
iona
l, ta
rget
ed a
ltera
tion
thro
ugh
site
-dire
cted
mut
agen
esis
Sm
all c
hang
es to
a D
NA
seq
uenc
e (~
1-5
nt)
1.D
esig
n co
mpl
emen
tary
prim
ers
with
alte
red
DN
A s
eque
nce
(can
be
spec
ific
sequ
ence
or r
ando
miz
ed s
eque
nce)
2.P
erfo
rm P
CR
on
entir
e pl
asm
id3.
Dig
est p
aren
t pla
smid
with
rest
rictio
n en
zym
e th
at ta
rget
s m
ethy
late
dD
NA
4.Tr
ansf
orm
into
app
ropr
iate
hos
tC
an p
erfo
rm o
n fra
gmen
t and
follo
w w
ith c
loni
ng
Dire
cted
evo
lutio
n
Ran
dom
mut
agen
esis
Per
form
PC
R u
nder
con
ditio
ns th
at in
crea
se th
e m
utat
ion
rate
of t
he p
olym
eras
e
Rec
ombi
natio
n **
**
**
* **
* **
**
fam
ily o
f re
late
d se
quen
ces
-ran
dom
dig
estio
n of
frag
men
ts a
nd
asse
mbl
y-d
irect
ed c
ross
over
eve
nts
and
asse
mbl
y
Pul
ling
out f
unct
iona
l mem
bers
of l
arge
libr
arie
s
-sel
ectio
n: a
n as
say
that
link
s th
e de
sire
d pr
oper
ty o
f the
mol
ecul
e to
cel
l gr
owth
-scr
een:
an
assa
y th
at li
nks
the
desi
red
prop
erty
of t
he m
olec
ule
to a
det
ecta
ble
outp
ut s
igna
l
Cha
ract
eriz
atio
n To
ols
DN
Am
RN
Apr
otei
n
transcription
translation
decay
AB
Pur
ifica
tion
of
cellu
lar c
onst
ituen
ts-D
NA
-RN
A-p
rote
ins
-met
abol
ites
mR
NA
Nor
ther
n bl
ot a
naly
sis
Rea
l-tim
e qu
antit
ativ
e P
CR
(qR
T-P
CR
)
run
RN
A o
ut o
n ge
ltra
nsfe
r RN
A to
m
embr
ane
hybr
idiz
e w
ith
labe
led
prob
e fo
r tra
nscr
ipt o
f in
tere
st
dete
ct a
nd
quan
tify
the
prob
e si
gnal
mR
NA
cDN
A
mak
e cD
NA
copi
es o
f the
RN
Aam
plify
regi
on o
f the
cD
NA
quan
tify
ampl
ifica
tion
durin
g P
CR
cycl
e nu
mbe
r
signal
**
*
Diff
eren
ces
in re
lativ
e st
artin
g am
ount
s of
cD
NA
tem
plat
e ar
e re
flect
ed in
diff
eren
ces
in c
ycle
num
bers
whe
n ex
pone
ntia
l pha
se
of a
mpl
ifica
tion
is o
bser
ved
mR
NA
Mic
roar
rays
mR
NA
cDN
A
mak
e cD
NA
copi
es o
f tot
al R
NA
fluor
esce
ntly
labe
l cD
NAs
*
Mic
roar
ray
met
hods
hav
e be
en a
dapt
ed to
det
ectin
g pr
otei
ns a
nd m
etab
olite
s
hybr
idiz
e to
a c
hip
with
co
mpl
emen
tary
ssD
NA
spot
ted
in a
rrays
Pro
tein
sW
este
rn b
lot a
naly
sis
Ana
lytic
al te
chni
ques
run
prot
ein
out
on g
eltra
nsfe
r pro
tein
to
mem
bran
ehy
brid
ize
with
la
bele
d pr
obe
to
prot
ein
of in
tere
st
(ant
ibod
y)
dete
ct a
nd
quan
tify
the
prob
e w
ith a
sec
onda
ry
antib
ody
Com
mon
ly u
sed
–liq
uid
chro
mat
ogra
phy-
mas
s sp
ectro
met
ry (L
C-M
S),
tand
em
MS
-MS
, 2-d
imen
sion
al g
el e
lect
roph
ores
is (2
D-G
E)
MS
is u
sed
for i
dent
ifica
tion
LC, g
as c
hrom
atog
raph
y, G
E, a
nd c
apilla
ry e
lect
roph
ores
is a
re u
sed
to
sepa
rate
and
pur
ify
Act
ivity
and
repo
rter a
ssay
s
Rep
orte
r enz
ymes
Mos
t com
mon
ly u
sed
is β
-gal
acto
sida
se(e
ncod
ed b
y th
e la
cZge
ne)
Plat
e-ba
sed
assa
y
+
yello
w
Solu
tion
/ qua
ntita
tive
assa
y
Oth
er c
omm
only
use
d re
porte
r enz
ymes
are
luci
fera
ses
Act
ivity
and
repo
rter a
ssay
s
Rep
orte
r pro
tein
s
Fluo
resc
ent r
epor
ter p
rote
ins
are
com
mon
ly u
sed
and
requ
ire n
o su
bstra
te
FRET
app
licat
ions
GFP
was
orig
inal
fluo
resc
ent r
epor
ter p
rote
inR
esea
rche
rs h
ave
spen
t sig
nific
ant e
ffort
gene
ratin
g: c
olor
-shi
fted
varia
nts,
mor
e ra
pidl
y fo
ldin
g an
d de
grad
ing
varia
nts,
mon
omer
icfo
rms,
brig
hter
fluo
resc
ence
, res
ista
nt to
ph
otob
leac
hing
, opt
imiz
ed fo
r diff
eren
t org
anis
ms
Act
ivity
and
repo
rter a
ssay
s
Use
of f
luor
esce
nt re
porte
r pro
tein
s as
read
outs
of c
ellu
lar c
ompo
nent
s an
d ne
twor
ks
Prom
oter
fusi
on
Prot
ein
fusi
on
Ope
ron
fusi
on
GFP
P PG
FP
PG
FP