Reverse Iranscriplase
+ 5-CH 3
dNTPs First-strand
Second·strand
cDNA
synthesis:
1st
phase:
ANase
H
+
DNA
Polymerase
I
+dNTI's
2nd
phase:
T4
DNA
Polymerase
I
+dNTI's
CH3C>t30i3
Oligo
(dT)
primer/linker
Mature
transcript
5'~3'
cDNA
+
- Mature
CJ'CL1!GlTI'ITl
~
Of:3CH30t3
Doubl
e·stranded
cDNA
~
~
~
-
3
' AA»AAAA
-
-
-
s· transcript
~
--EcoRI
Adapters
+
T
4 DNA
Ligase
Xhol
restrtctlon
digestlon
+
rn 3
CH 3
CH
3
size
fractionation
Xhol CH 3
rn 3
rn 3
EcoRI
1\1Q'JC ~C'KG'GITITITlT
~
G
Double·stranded
cDNA
G
L______j ~
L______j Cl'D\1\ prepared
to
be
cloned
'I'CG'GITITITIT
~
~ G C1W>JW>AA
-
L______j CTlN\
Stuffer
sequf
nce
Vector
restriction
Anns
annealing
CAnn
< - cos
Vector
cos
~
­
+
<=3:
+
<::::r
Figure
38
Cloning
Method
Uslng
Lamb
da
Bacterlophage
Vector.
Growth
on lnfection
of
plates
as Llgation
Packaging~
~
~ hast
cell
plaques
<
(
<
=
=
= 0
0
<(jfff5C
<
<
-:::::
:::::
::::: -
o
o
-
oNA
Insert
~
N
"-J
0\
t"'
5 N
:n 0
z
c: (')
:z:
() >
z
tl G'
l
::d
tT1
G'
l
0
::d
>< ~ G'
l
tT1
::d
~
z 0