Simple Cloning and DNA Assembly in Escherichia coli by Prolonged Overlap Extension PCR - DNA Cloning and Assembly Methods - page 184

a

Vector

IF1

Insert 1

IR1

IF2

Insert 2

IR2

VF

VR

QC1 QC2 QC3

b

1 2 3 4 5 6 7 M

kb

10

8

6

5

4

3.6 kb

3

2

1.5

1

768 bp

0.5

258 bp

Fig. 4 DNA assembly of three fragments. Lane 1 PCR linearized pET20b vector,
Lane 2 PCR linearized fbp insert, Lane 3 PCR linearized rfdoc insert, Lane 4 DNA
multimers generated by prolonged overlap extension PCR, Lane 5 DNA multim-
ers digested with NdeI and XhoI, Lane 6 a purifi ed plasmid from a randomly
selected colony, Lane 7 a purifi ed plasmid digested with NdeI and XhoI; and M, a
1-kb DNA ladder from NEB

8. Three microliters of POE-PCR product was examined by
0.8 % agarose gel electrophoresis (Fig. 4b , lane 4). To check
whether the two inserts were successfully incorporated into the
plasmid backbone, the POE-PCR product can be digested by
the restriction enzymes (Fig. 4b , lane 5).
9. Directly transform the POE-PCR product to competent E . coli
cells and spread them on plate.