TAKARA CycleavePCR VT1 User Manual Download

Page: 1 / 17

TAKARA BIO INC.

URL:http://www.takara-bio.com

CycleavePCR

O157 (VT1/VT2) Detection Kit Ver.2.0

v.0607

Cat.#CY203

Table of contents

I. Description...............................................................................2

II. Kit Components.........................................................................3

III. Reagents and Instruments Required..........................................3

IV. Storage......................................................................................3

V. Cautions....................................................................................4

VI. Protocols....................................................................................4

VI-1. Sample preparetion......................................................5

VI-2. Setting of Smart Cycler

II System...............................5

VI-3. Reaction mixture ........................................................7

VI-4. Display of results........................................................9

VI-5. Judgement..................................................................11

VII. Trouble Shooting......................................................................15

VIII. Reference.................................................................................16

IX. Related products.......................................................................17

Summary of Contents for CycleavePCR VT1

Page 1: ...ntents I Description 2 II Kit Components 3 III Reagents and Instruments Required 3 IV Storage 3 V Cautions 4 VI Protocols 4 VI 1 Sample preparetion 5 VI 2 Setting of Smart Cycler II System 5 VI 3 Reaction mixture 7 VI 4 Display of results 9 VI 5 Judgement 11 VII Trouble Shooting 15 VIII Reference 16 IX Related products 17 ...

Page 2: ...ys Cycling Probe Technology CPT 2 for detection which is a high sensitive detection method utilizing a combination of chimera probe composed of RNA and DNA and RNase H The specific sequence of target gene to be amplified can be detected efficiently during or after amplification by this method The 5 end of the probe is labeled with a fluores cent substance and the 3 end is labeled with a quencher w...

Page 3: ...e light shielding environment II Kit Components 25 µl 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 RNa a a as s s se e e eH 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 amplificati...

Page 4: ...n Area 3 Addition of sample into a reaction mixture and perform reaction and detection As this kit performs amplification and detection simultaneously through real time PCR there is no need to use amplified product obtained from the reaction to subsequent process such as eletrophoresis etc Please refrain from taking amplified products out of tubes It can result in contamination V Cautions VI Proto...

Page 5: ...d culture solution should be prepared by following the standard protocol appropriate for each food sample Heat extracted sample can be stored at 20 C For more information on handling Smart Cycler II System see the instructions supplied with it 1 Start the Smart Cycler II System 2 Set the protocol Click the icon Define Protocols and then New Protocol button to create the protocol by following the s...

Page 6: ...ave been set under a name FAM at initialization no entry is required here 3 2 Set the amplification curve TET for the amplified product derived from Internal Click the icon Define Graphs and create the graphs by following the steps shown below Since the graphs have been set under a name TET at initialization no entry is required here 3 3 Set the amplification curve ROX for the amplified product de...

Page 7: ...rotocol and Sites screen This product detects simultaneously three amplification products VT1 VT2 and internal control within one reaction tube In order to obtain the right detection result we recommend you to perform VT1 positive control reaction VT2 positive control reaction and a negative control reaction altogether 1 Prepare the reaction mixture on ice shown below Perform in Area 1 Prepare the...

Page 8: ... positive control add 1 µl as template Add the components of the above reaction mixture without template into a fresh tube by pouring on the tube wall Please refer to the following figure Close gently the lid of the tubes not so tightly and move to the Area 3 2 Addition of sample template Perfom in Area 3 Prepare one tube of negative control by adding sterilized distilled water instead of sample F...

Page 9: ...nd Temperature the temperature chart graph Click and show the Select Graphs When the graphs for FAM TET ROX and Temperature have been selected at the initial setting no entry is required here Choose the site and protocol to be used and click the OK button 3 From the Views list select Results Table and enter Sample ID Click the Results Table Enter sample ID 4 Click Analysis Settings for opening Cli...

Page 10: ...T1 VT2 Detection Kit Ver 2 0 v 0607 Cat CY203 URL http www takara bio com 5 Select the amplified curve of interest in the above Views Amplified curve for FAM VTI gene Amplified curve for TET Internal control Amplified curve for ROX VT2 gene ...

Page 11: ...e PCR process POS is displayed in the cell of the FAM Std Res the results of detection of the fluorescent signal derived from VT1 gene in the Results Table while if it is smaller than 100 NEG is displayed If the fluorescent signal value for ROX detection is 100 or larger POS is displayed in the cell of ROX Std Res the detection result of the fluorescent signal derived from VT2 gene in the Results ...

Page 12: ...ternal control ROX Std Res whichever it is POS or NEG It was displayed on FAM Std Res as OOO in VT1 positive control Internal control is not amplified either There is a certain problem about the whole reaction sys tem and reaction and detection are not per formed normally Reform the reaction again There is no problem about VT1 detection system VT1 gene in a sample is below a detec tion limit There...

Page 13: ...is not amplified either There is a certain problem about the whole reaction sys tem and reaction and detection are not per formed normally Reform the reaction again Displayed as POS Displayed as NEG There is no problem about VT2 detection system VT2 gene in a sample is below a detec tion limit There is a problem about VT2 detection system A problem is in primer for VT2 amplifica tion or in a detec...

Page 14: ...blem 4 in VT1 detection Judgement impossible 3 TxR POS VT2 contamination suspected 6 VT2 contamination suspected 6 Std Res VT2 NEG No VT2 contamination 2 Judgement impossible 3 Table 3 VT2 positive control reactions VT1 negative control TET Std Res International control POS NEG FAM POS VT1 contamination suspected 6 VT1 contamination suspected 6 Std Res VT1 NEG No VT1 contamination 2 Judgement impo...

Page 15: ...trol may be degraded 5 It is suspected that there may be problem in Primers for VT2 amplification or in Probe for VT2 detection or that VT2 Positive Control may be degraded 6 Contamination is suspected Retry after decontamination of the place where the reaction mixture is prepared and the equipment used VII Trouble shooting 1 A numeric value appears in the FAM TET or TxR Std Res column instead of ...

Page 16: ... After the Y axis Max value was manually adjusted to 500 VIII Reference 1 Takao T T Tanabe Y M Hong Y Shimonishi H Kurazono T Yutsudo C Sasakawa M Yoshikawa and Y Takeda 1988 Identity of molecular structure of Shiga like toxin VT1 from Esherichia coli O157 H7 with that of Shiga toxin Microb Pathog 5 357 369 2 Jackson M P R J Neil A D O Brien R K Holmes and J W Newland 1987 Nucleotide sequence anal...

Page 17: ...rcial consideration is hereby granted by implication or estoppel Further information on purchasing licenses to practice the PCR process where the process is covered by patents may be obtained by contacting the Director of Licensing Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 or the Licensing Department Roche Molecular Systems Inc 1145 Atlantic Avenue Alameda California...

Search results

Summary of Contents for CycleavePCR VT1

Reviews:

Related manuals for CycleavePCR VT1

Brands by name

Popular brands

TAKARA CycleavePCR VT1, Manual

Search results

Summary of Contents for CycleavePCR VT1

Reviews:

Related manuals for CycleavePCR VT1

Brands by name

Popular brands