3
TAKARA BIO INC.
URL:http://www.takara-bio.com
CycleavePCR
TM
O157 (VT1/VT2) Detection Kit Ver.2.0
v.0607
Cat.#CY203
1.
TaKaRa Ex Taq
TM
R-PCR 5 units/
µ
l 12.5
µ
l (50 reactions)
2. Tli RNase H II*
1
200 units/
µ
l 25
µ
l (50 reactions)
3. 5 X Reaction Mixture*
2
5 X conc. 250
µ
l (50 reactions)
4. VT Primer Mix*
3
5
µ
M each 100
µ
l (50 reactions)
5. VT Chimera Probe Mix*
4
5 X conc. 250
µ
l (50 reactions)
6. VT1 Positive Control 1 X 10
4
copies/
µ
l 10
µ
l (10 reactions)
7. VT2 Positive Control 1 X 10
4
copies/
µ
l 10
µ
l (10 reactions)
8. distilled water
1 ml
*1 Tli RNase H II is a thermostable RNase H derived from
Thermococcus litoralis
.
*2 Including dNTP Mixture and Internal Control.
*3 Primers are manufactured by SHIMADZU CORPORATION.
*4 Including probe for control reaction. Be sure to store the fluorescent labeling probes in the
light-shielding environment.
II. Kit Components (25
µ
l X 50 reactions):
Q
F
キメラ
probe
増幅産物
RNA
Q
F
ハイブリッド形成
Q
F
RNaseH
による
RNA
部分の切断
Q
F
蛍光強度増大
蛍光物質
クエンチャー
RN
aaaasssseeeeH
H
H
H
Fluorescent Quencher
substance
Chimeric probe
Amplified products
Formation of hybrid
Cut RNA part by RNaseH
Increase of fluorescence intensity
Principle of ALDH2 typing:
[ Probe labeling, a detection channel, and amplification size ]
Probe fluorescence substance Detection channel Amplification size
VT1 FAM and Quencher Ch1 171 bp
VT2 ROX and Quencher Ch3 171 bp
Internal control TET and Quencher Ch2 110 bp
III. Reagents and Instruments Required but Not Supplied in the Kit:
IV. Storage: -20
°
C (for shipping and storage)
[Reagents]
Sterilized distilled water
[Equipment] 1. Smart Cycler
®
II System (Real time PCR instrument)
(Cepheid)
2. Special tubes for Smart Cycler
®
3. Desk-top centrifuge for Smart Cycler
®
4. Heat block (applicable at 95
°
C)
[Others]
1. Micropippets for 200
µ
l, 20
µ
l and 10
µ
l.
2. Micropippet tips (with hydrophobic filter)