QuantiTect Probe RT-PCR プロトコールとトラブルシューティング ( /2008)

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1 January 2008 QuantiTect Probe RT-PCR 1 RT-PCR Applied Biosystems Cycler 1 RT-PCR 2 LightCycler 1.x RT-PCR 5 9 Sample & Assay Technologies

2 Applied Biosystems Cycler 1RT-PCR TaqMan Applied Biosystems Bio-Rad /MJ Research Cepheid Corbett Eppendorf Roche Stratagene LightCycler 1.x LightCycler µl Applied Biosystems 7500 Fast SystemSmartCycler system 25 µl LightCycler µl RT mix 2x QuantiTect Probe RT-PCR Master Mix QuantiTect RT Mix 1/100 UNG 1 RT-PCR bp RT-PCR PCR HotStarTaq DNA Polymerase cdna 50 µl 2x QuantiTect Probe RT-PCR Master Mix QuantiTect RT Mix threshold 2x QuantiTect Probe RT-PCR Master Mix dutp uracil-n-glycosylase UNG UNG Applied Biosystems 7500 threshold QuantiTect Probe RT-PCR 01/2008

3 1. 2x QuantiTect Probe RT-PCR Master Mix 20 RNA RNase QuantiTect RT Mix µl 2x QuantiTect Probe RT-PCR Mix QuantiTect RT Mix 2x QuantiTect Probe RT-PCR Master Mix 4 mm Mg 2+ Mg 2+ 6 mm 1. 2x QuantiTect Probe RT-PCR Master Mix* 25 µl 1x A 0.4 µm B 0.4 µm µm QuantiTect RT Mix 0.5 µl RNA 4 1 pg 500 ng RNase 2 unit Uracil-N-glycosylase UNG 50 µl * 4 mm MgCl 2 50 µl 2x master mix 2x master mix µl= 0.5 xµl 0.4 µm 0.4 µm 1 µm 50 µlrt mix RT mix µl = 0.01 xµl QuantiTect Probe RT-PCR 01/2008 3

4 3. PCR 4. RNA 1 pg 500 ng PCR 5. 2 UNG 5 6. PCR 2. * RNA 55 PCR HotStarTaq DNA Polymerase 2 * RNA * SmartCycler 1 4 QuantiTect Probe RT-PCR 01/2008

5 LightCycler 1.x 2.0 1RT-PCR LightCycler 1.x LightCycler 2.0 FRET TaqMan 2 RT-PCR bp RT-PCR PCR HotStarTaq DNA Polymerase cdna 20 µl 2x QuantiTect Probe RT-PCR Master Mix QuantiTect RT Mix second derivative maximum fit-point noise band 2x QuantiTect Probe RT-PCR Master Mix dutp uracil-n-glycosylase UNG UNG 1. 2x QuantiTect Probe RT-PCR Master Mix 20 RNA RNase QuantiTect RT Mix µl 2x QuantiTect Probe RT-PCR Master Mix QuantiTect RT Mix 2x QuantiTect Probe RT-PCR Master Mix 4 mm Mg 2+ Mg 2+ 6 mm QuantiTect Probe RT-PCR 01/2008 5

6 3. 2x QuantiTect Probe RT-PCR Master Mix* 10 µl 1x A 1 µm B 1 µm 0.2 µm QuantiTect RT Mix 0.2 µl RNA 4 1 pg 1 µg RNase 2 unit Uracil-N-glycosylase UNG 20 µl * 4 mm MgCl 2 1 µm 0.5 µm 2 µm 3. PCR 4. RNA 1 pg 1µgPCR UNG FRET 5 TaqMan LightCycler 6 6. PCR LightCycler 6 QuantiTect Probe RT-PCR 01/2008

7 4. FRET / RNA 55 PCR / HotStarTaq DNA Polymerase / / T m / RNA QuantiTect Probe RT-PCR 01/2008 7

8 5. TaqMan / RNA 55 PCR / HotStarTaq DNA Polymerase / / RNA 6. FAM F1 F1/1 F1/F2 F2 F2/1 F2/F1 LC-Red 640 F3 F3/1 F3/F1 LC-Red QuantiTect Probe RT-PCR 01/2008

9 PCR a FRET Molecular Beacons b FRET Molecular Beacon c d e HotStarTaq DNA Polymerase f HotStarTaq DNA Polyerase g LightCycler 1.x LightCycler 1.x FRET Molecular Beacon RNA * RT-PCR HotStarTaq DNA Polymerase 9515 HotStarTaq DNA Polymerase LightCycler 1.x * PCR QuantiTect Probe RT-PCR 01/2008 9

10 h QuantiTect RT Mix i j QuantiTect RT Mix QuantiTect Probe RT-PCR Master Mix RT-PCR k l m Mg 2+ n QuantiTect RT Mix LightCycler µl RT mix LightCycler 1.x µl RT mix SmartCycler Applied Biosystems 7500 Fast System 0.25 µl RT mix 0.5 µl RT mix QuantiTect RT Mix QuantiTect Probe RT-PCR Master Mix QuantiTect RT Mix RT-PCR bp RT-PCR 300 bp RT-PCR RT-PCRprimer design guideline * LightCycler 1.x µm 0.4 µm 2 µm * 2x QuantiTect Probe RT-PCR Master Mix Mg 2+ 4 mm Mg 2+ 6 mm 0.5 mm RNA * RNA RT-PCR * PCR 10 QuantiTect Probe RT-PCR 01/2008

11 o p q r s t u v w x UNG RNA * Molecular Beacons probe design guideline DNase I Molecular Beacon DNase I RT-PCR RT-PCR * RT-PCR UNG UNG UNG 50 cdna * PCR QuantiTect Probe RT-PCR 01/

12 LightCycler1.x 2.0 y LightCycler 1.x 2.0 z C T Crossing point a LightCycler 1.x µg 500 ng b c QuantiTect RT Mix QuantiTect RT Mix LightCycler µl RT mix LightCycler 1.x µl RT mix SmartCycler Applied Biosystems 7500 Fast System 0.25 µl RT mix 0.5 µl RT mix No Template a b F1 FAM TaqMan Molecular BeaconsF2 LC-Red 640FRET F3 LC -Red 705FRET uracil-n-glycosylase 12 QuantiTect Probe RT-PCR 01/2008

13 No Reverse Transcription DNA RNA a b c ABI PRISM 7000 d 25 µl optical adhesive cover 50 µl 3 2,000 LightCycler 1.x 2.0 e RT-PCR f g cdna DNA RNA DNase Analysis Setting RT-PCR LightCycler QuantiTect Probe RT-PCR 01/

14 Memo 14 QuantiTect Probe RT-PCR 01/2008

15 Trademarks: QIAGEN, HotStarTaq, QuantiTect (QIAGEN Group); ABI PRISM, Applied Biosystems, FAM (Applera Corporation or its subsidiaries); Bio-Rad (Bio-Rad Laboratories, Inc.); Eppendorf (Eppendorf AG); TaqMan, Roche, LightCycler (Roche Group); Cepheid, SmartCycler (Cepheid); Stratagene (Stratagene). NOTICE TO PURCHASER: LIMITED LICENSE A license to perform the 5' nuclease process for research requires the use of a Licensed 5' Nuclease Kit (containing Licensed Probe), or the combination of an Authorized 5' Nuclease Core Kit plus Licensed Probe, or license rights that may be purchased from Applied Biosystems. This product (QuantiTect Probe RT-PCR Kit) is an Authorized 5' Nuclease Core Kit without Licensed Probe. Its purchase price includes a limited, non-transferable immunity from suit under U.S. Patents Nos. 5,210,015, 5,487,972, 5,476,774, and 5,219,727, and corresponding patent claims outside the United States, owned by Roche Molecular Systems, Inc. or F. Hoffmann-La Roche Ltd (Roche), for using only this amount of the product in the practice of the 5' nuclease process solely for the purchaser s own internal research when used in conjunction with Licensed Probe. This product is also an Authorized 5' Nuclease Core Kit for use with service sublicenses available from Applied Biosystems. This product conveys no rights under U.S. Patents Nos. 5,804,375, 6,214,979, 5,538,848, 5,723,591, 5,876,930, 6,030,787, or 6,258,569, or corresponding patents outside the United States, expressly, by implication or by estoppel. No right under any other patent claims (such as apparatus or system claims in U.S. Patent No. 6,814,934) and no right to perform commercial services of any kind, including without limitation reporting the results of purchaser's activities for a fee or other commercial consideration, is hereby granted expressly, by implication or by estoppel. This product is for research use only. Diagnostic uses require a separate license from Roche. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA. NOTICE TO PURCHASER: LIMITED LICENSE The purchase price of this product (QuantiTect Probe RT-PCR Kit) includes a limited, non-transferable license under U.S. Patents Nos. 5,407,800, 5,322,770, 5,310,652 and corresponding patent claims outside the United States, owned by Roche Molecular Systems, Inc. or F. Hoffmann-La Roche Ltd (Roche), to use only this amount of product solely for the purchaser s own internal research. No right under any other patent claims (such as apparatus or system claims) and no right to use this product for any other purpose or for commercial services of any kind, including without limitation reporting the results of purchaser s activities for a fee or other commercial consideration, is hereby granted expressly, by implication or by estoppel. This product is for research use only. Diagnostic uses require a separate license from Roche. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA. NOTICE TO PURCHASER: LIMITED LICENSE. This product (QuantiTect Probe RT-PCR Kit) or its use is covered by at least one claim of U.S. Pat. Nos. 5,035,996; 5,945,313; 6,287,823; or 6,518,026, owned by Invitrogen Corporation. The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer (whether the buyer is an academic or for-profit entity). The buyer cannot sell or otherwise transfer (a) this product, (b) its components, or (c) materials made by the employment of this product or its components to a third party or otherwise use this product or its components or materials made by the employment of this product or its components for Commercial Purposes. Commercial Pur-poses means any activity for which a party receives or is due to receive consideration and may include, but is not limited to: (1) use of the product or its components in manufacturing; (2) use of the product or its components to provide a service, information, or data; (3) use of the product or its components for therapeutic, diagnostic or prophylactic purposes; or (4) resale of the product or its components, whether or not such product or its components are resold for use in research. The buyer cannot use this product or its components or materials made using this product or its components for therapeutic, diagnostic or prophylactic purposes. Further information on purchasing licenses under the above patents may be obtained by contacting the Licensing Department, Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, CA outlicensing@invitrogen.com. QIAGEN QIAGEN, all rights reserved.

16 Forefront Tower II Tel: Fax: /2008 Sample & Assay Technologies

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Unknown Cell Sorting S3e EVOLUTION 1 STREAMLINED 2 PRINCIPLE 3 HIGH PERFORMANCE 4 AUTOMATION 5 6 INNOVATIVE 7 8 VERSATILE 9 INTUITIVE 10 BIO SAFETY 11 BioSafety Air in Air out Air in Air in System airflow 12 SOLUTIONS