Biomedical Engineering Reference
In-Depth Information
1
st
expression
cassette in
pACYCDuet-1
2
nd
expression
cassette in
pACYCDuet-1
vector
specific
vector
specific
B specific
A specific
PCR
amplification
PCR
amplification
5
'
3
'
5
'
3
'
3
'
5
'
3
'
5
'
A = Az or TFIIE
α
B = ODC or TFIIE
β
b
pT7/lac
gene A
DuetUP2
pACYCDuetUP1
pT7/lac
DuetDOWN1
pACYCDuet-
His-gene A/gene B
pACYCDuet-1
PetRev
Fig.
1
Schematic representation of the strategy for simultaneous RF cloning. (
a
) Gene A and gene B are shown
in
black
and
red lines
, respectively. Primers are indicated by
arrows
containing gene-specifi c sequences
(marked in the same color as the gene) and vector-specifi c sequences indicated by
dotted lines
. The vector-
specifi c sequences fl anking gene A are shown as
red squares
and
purple circles
, whereas for gene B, the
sequences are shown as
blue squares
and
green circles
. Gene A represents in our study Antizyme (Az) or
transcription factor TFIIE
α
(TFIIE
α
) and gene B represents Ornithine Decarboxylase (ODC) or transcription fac-
tor TFIIE
). (
b
) Schematic representation of integration of gene A and gene B into the two expression
cassettes in the expression vector pACYDuet-1. Color coding of the genes and fl anking regions are as in (
a
).
Arrows
indicate primers used in colony PCR screening (
see
Fig.
2
and Table
1
) (reproduced from ref.
9
with
permission from Elsevier) (color fi gure online)
β
(TFIIE
β
Component
Stock solution
Volume/50
μ
L reaction
Final concentration
Target DNA
10 ng/
μ
L
2
μ
L
0.4 ng/
μ
L
Forward primer
25
μ
M
1
μ
L
0.5
μ
M
Reverse primer
25
μ
M
1
μ
L
0.5
μ
M
μ
μ
dNTPs mix
10 mM each
1
L
200
M each
Phusion HF Buffer
5×
10
μ
L
1×
Phusion DNA Polymerase
2 U/
μ
L
0.8
μ
L
0.032 U/
μ
L
Ultrapure water
34.2
μ
L
