Simple Cloning and DNA Assembly in Escherichia coli by Prolonged Overlap Extension PCR - DNA Cloning and Assembly Methods - page 180

Vector

IF VF

5'-TTAAC···TTCAT ATGGT···GGATA-3'

5'-CAAGC···TAAGC TGTGG···AACTG-3'

CAAGC···TAAGC TGTGG···AACTG
GTTCG···ATTCG ACACC···TTCAC

TTAAC···TTCAT ATGGT···GGATA

Gene of interest

AATTG···AAGTA TACCA···CCTAT

3'-GTTCG···ATTCG ACACC···TTCAC-5'

3'-AATTG···AAGTA TACCA···CCTAT-5'

IR

VR

Vector-specific
sequence (20-25nt)

Gene-specific
sequence (20-25nt)
Fig. 1 Primer design for Simple Cloning that can insert one DNA fragment into any place of plasmid

Vector template Insertion template 1

Insertion template 2

PCR

PCR PCR

Purification Purification

Purification
Vector backbone

QC1 Insert 1 QC1 QC1

Insert 2

POE-PCR

DNA multimers QC2

Direct transformation

Colonies

Plasmid extraction

Plasmids QC3

Fig. 2 Flow schemes of Simple Cloning and DNA assembly by POE-PCR. First,
two or three or four 3 overlapped insert and vector fragments are gener-

ated by regular PCR. Second, DNA multimers are formed in vitro by prolonged
overlap extension PCR. Third, E . coli strains can cleave DNA multimers to a cir-
cular plasmid, the desired chimeric plasmid. QC means quality control

′ and 5 ′

or GC Buffer ( see Note 3 ); and the Phusion DNA polymerase,
0.02 U

µL. The PCR program is 98 °C denaturation, 30 s; 30
cycles of 98 °C denaturation, 15 s; 55 °C annealing ( see Note 4 ),
15 s; extension at 72 °C at 3 kb per min for the targeted frag-
ment; and 5 min extension at 72 °C. The quality of the PCR
product can be checked by examining 3

/

L of the product by
0.8 % agarose gel electrophoresis (Fig. 3a , lane 1).
2. The insert fragment is amplifi ed with a pair of primers of IF
and IR through PCR by using the Phusion DNA polymerase.

μ