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1、500
pMD18-T Vector
D101A
1 pg
500
pMD19-T Vector
D102A
31 • BIO VIEW No2
pMD18-T Vector pMD19-T Vector pUC18 pUC19
PCR
(TA Cloning)
2、)
Control Insert (500 bp)
Control
pMD19-T Vector
pMD18-T Vector
pMD19-T Vector
PMD18-T & pMD19-T Vector
pMD18-T Vector
flcaBEST1M Sequencing Primer RV M
GAGCGGATAACAATTTCACACAGG
>9cZ
X/nai
ECOR V
「Cloning Site
Hine 11
Kpni\ IBamH I Xbn fcoR \ Sall \ Psti
1 i i
3、 I i i i i i i I i
Accl Sse6387 I
Sph! Hind ill
GGCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCG
CAGCACTGACCCTTTTGGGACCGC
BcaBEST " Sequencing Pnmer M13-47
Digested by EcoR v
pMD19-T Vector
—t c t agaga t T a t eg t cgacc -- ..-agatet c ta Ttagcagc tgg --
(T CIorwg Site)
BcaBEST™ Seq
4、uencing Primer RV M
GAGCGGATAACAATTTCACACAGG ► Hine II
S$e8387 I Acc t X/m I
WZ ► Hind III |~Psn I Si/1 EcoR V Xbf I 严mH 打 Se屮卄 Stci £coR I
GAGCGGATAACAATTTCACACAGG AAACAGCTATGACCATGATTACGCCAAGCTTGCATGCCTGCAGGTCGACGATATCTCTAGAGGATCCCCGGGTACCGAGCTCGAATTC
Digested by EooR V
ACTGGCCGTCGTTTTACAA
5、CGTCGTGACTGGGAAAACCCTGGCG
CAGCACTGACCCTTTTGGGACCGC
BcaBEST^ Sequencing Primer Ml 3-47
…gtcgacgatT ・・・caget gc ta
atetetaga...
Ttagagatct... ("lomng Site)
10 pl 1
30 pl 5
pMD18-T orpMD19-T Vector (50 ng/pl)
Control Insert (50 ng/pl)
Ligation Mix*
BIO VIEW no.2 • 32
6、B.
Control
Insert
(efu/pg Vector)
(%)
(%)* L W
2.8 10°
96.3
90
2.2 10、
40.2
0
Bea
Cloning site
419
BcaBEST Sequencing Primer M13-47 binding site 347 ~ 370
BcaBEST Sequencing Primer RV-M binding site
478~ 500
LacZ operator
146 ~ 469
ColE1 ori
7、
852* 1466
Amp
1626-2486
Cloning site
431
BcaBEST Sequencing Primer M13-47 binding site 347 ~ 370
DcaD匚ST Sequencing Primer RV-M binding site
478 - 500
LacZ operator
146 ・ 469
ColE1 ori
852~1466
Amp
1626 ~ 2486
A.
P
pMD1 &T or pMD19-T Vector*1
Control Insert*2
dH2O
3gl
pM
8、D18-T or pMD19-T Vector* 1
1
Insert DNA*3
0.1 pmol ~ 0.3 pmol
dHzO
up to 5 pl
LacZ
LacZ a
P
a
33 • BIO VIEW No2
Q&A
TaKaRa
Pyrobest Pfx Pfu
Bea
Q-2
A-2
Q-3
A-3
Q-4
A-4
LacZ
pMD1819T载体图谱
