•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