実験テキストのPDFファイルは - 千葉工業大学工学部｜生命環境科学科

5S 生命環境科学科
遺伝子工学実験テキスト
平成 2６年度版
―担当教員―
坂本泰一
河合剛太
滝口泰之
根本直樹
黒崎直子
1
2
3
4
5
6
7
8
9
10
I-1
I-2
I-3
I-4
I-5
RNA
RNA
RNA
HIV-1
RNA
DIS
RNA
PAGE
①
RNA
②
RNA
③
RNA
PAGE
①
②
③
①
②
HIV-1
RNA
II-1
39
DIS
39
RNA
2
RNA
Structural
Requirement
Immunodeficiency
Virus
for
the
Type-1
Two-Step
Genome,
Dimerization
Takahashi,
K.,
of
Human
Baba,
S.,
Chattopadhyay, P., Koyanagi, Y., Yamamoto, N., Takaku, H., and Kawai, G.,
RNA 6, 96-102 (2000).
II-2
,
(MIC)
.
2.1.
,
.
,
,
,
,
.
2.2.
2.2.1.
(
,
1 ml) ,
,
, 300 ml
10 ml
, L
,
,
,
, BTB
,
,
,
(
,
,
, 100 ml
,
,
L
,
660 nm),
,
2.2.2.
,
12
70
,
,
,
,
,
,
,
Escherichia coli NBRC
,
3301
2.3
2.3.1.
phosphate buffered saline
100 ml
,
.
III-1
2.0 ml
PBS
6
, 5.0 ml
3
.
,
1 N HCl
pH
L
7
.
2
100 ml
7.0
7.2
, 1 N NaOH
BTB
8.9 ml
.
,
.
.
.
.
121
20
.
2.3.2.
10.0
5.0 ml
0.5 mg
,
PBS
,
,
(2000 µg/ml).
PBS 5.0 ml
160 µg/ml
2)
2.0 ml
.
PBS 2.0 ml
80 µg/ml
2
, 40
20 µg/ml
.
20
8.9 ml
160 µg/ml
1000 µl
,
2,4,8,16 µg/ml
.
,
8.9 ml
PBS 1000 µl
1
,
0 µg/ml
,
,
2000 µg/ml
1100 µl
1
8.9 ml
.
6)
5
.
L
L
660 nm
,
16 µg/ml
100 µl
5
0
L
,
5
.
37
.
L
,
.
,
30
660 nm
,
III-2
.
.
3
.
2.3.3.
,
121
20
,
1
.
, 70
.
G = 0.301(t
G
, N0
Nt
t0)/(log10Nt
log10N0)
t0
t
(cells/ml),
(cells/ml)
MIC
,
,
,
,
.
,
,
,
,
,
,
.
,
.
.
.
.
MIC
.
III-3
MIC
.
.
.
(1992).
17
(1992).
(1993).
(1985).
(1999).
KCl
0.20 g
KH2PO4
0.20 g
Na2HPO4
12H2O
2.90 g
NaCl
8.00 g
1000 ml
pH 7.0-7.2
2
3.0 g
10.0 g
NaCl
5.0 g
1000 ml
pH 7.0-7.2
III-4
,
.
,
, RNA
,
,
,
.
1
,
.
,
DNA
.
,
.
2.1.
P1
2.2.
2.2.1.
,
,
2.2.2.
50 mM
,
IV-1
LB
, LB
,
,
,
, DNA
,
DNA
(
/Hind
), 1%
(Escherichia coli DH5α
, TAE
)
2.3
2.3.1.
37
1.5 mL
LB
,
, 6,000 rpm, 4
,
1
ml
, 5
50 mM
,
, 6,000 rpm, 4
,
1
ml
100 µL
, 5
,
50 mM
, 6,000 rpm, 4
,
,
, 5
,
50 mM
2.3.2.
1 µL
(200 ng)
15
42
.
1
,
2
500 µL
.
LB
, 37
30
,
100 µL
LB
,
,
,
, 37
.
2.3.3.
1.5 mL
µL
,
10µL
(400 ng), 1 µL
1 µL
, 6 µL
10x
,
,
,
.
IV-2
, 2
,
2
37
,
.
30
2.3.4.
TAE
3 µL
100 V
,
30
DNA
,
pBR322
BamH
DNA
.
Sal
.
DNA
DNA
,
,
.
,
DNA
,
(108 cfu
)
IV-3
,
2012
2009
,
,
2009
2002
1 µg
pBR322
(colony forming unit : cfu)
,
,
,
, LB
,
.
/Hind
,
,
New England BioLabs (NEB)
.
1. NEB
https://www.neb.com/
2. Tools & Resources
, DNA
sequences and Maps tool
3.
4. Vectors
, Sites(pdf)
Map
, pACYC184,
pBR322, pUC19
.
1%
LB(Luria-Bertani)
LB
,
.
, 1000 mL
,
10 g,
5 g, NaCl 10 g.
,
.
IV-4
DNA
0.5X TAE
PCR
,
, Loading Dye,
100bpDNA
V-1
50g
1)
30
2)
DNA
10
3)
5
PCR
PCR
1)
PCR
µL
PCR
µL
2)
3)
PCR
PCR
1) 2.5%
2) PCR
Loading Dye
µL
3)
4)
5)
0.5X TAE
300
µL
6)
7)
400mL
100bpDNA
V-2
8) 100V
BPB
2/3
10
V-3

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