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Som abi7500 setup_e
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Som abi7500 setup_e

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  • 1. Technical Note ® Applied Biosystems 7500 (Fast block) Real-Time PCR Run Setup Instructions for qBiomarker Mutation PCR Arrays Before the Experiment  Please make sure the real-time PCR instrument is working properly. Refer to the manufacturer’s Installation and Maintenance manual if needed. Creation of PCR Protocol Template 1) 2) 3) Open the ABI 7500 v2.0.4 software on the desktop of the computer that is connected to the ABI 7500 instrument. Select File  New Experiment  Advanced Setup. Under Setup  Experimental Properties menu (See Figure 1) a) Select 7500 Fast (96 Wells). b) Select Quantitation – Standard Curve. c) Select Taqman Reagents. d) Select Standard (~ 2 hours to complete a run). Page 1 of 4 QIAGEN GmbH  QIAGEN Str. 1  40724 Hilden  Germany  Commercial Register Düsseldorf (HRB 45822) Managing Directors: Peer M. Schatz, Roland Sackers, Bernd Uder, Dr. Joachim Schorr, Douglas Liu
  • 2. Technical Note Figure 1: Experimental Properties 4) 5) Under Setup  Plate Setup menu a) In Define Targets and Samples tab, ensure that FAM is listed beside Target 1. For Quencher, select “none”. b) In Assign Targets and Samples tab 1) Highlight the entire plate in the View Plate Layout window. 2) Check the Assign box next to Target 1 under Assign targets to the selected wells and verify that all wells in Plate Layout view have the “U” symbol (“U” = unknown). Under Setup  Run Method menu (See Figure 2). a) Enter 25 µL for Reaction Volume Per Well b) Adjust parameters to reflect the following  Holding Stage  Temperature: 95°C  Time: 10:00  Cycling Stage (2 Steps)  Number of Cycles: 40  Step 1: 95°C, 00:15  Step 2: 60°C, 1:00  Detect and record FAM fluorescence from every well during the annealing step of each cycle. Page 2 of 4 QIAGEN GmbH  QIAGEN Str. 1  40724 Hilden  Germany  Commercial Register Düsseldorf (HRB 45822) Managing Directors: Peer M. Schatz, Roland Sackers, Bernd Uder, Dr. Joachim Schorr, Douglas Liu
  • 3. Technical Note Figure 2: Run Method 6) Select File Save As Template. Save the file as Experimental Template files (*.edt) with the filename “qBiomarker_Mutation_PCR_Array_Template” (click Save). Performing Real-Time PCR Detection 1) 2) 3) 4) 5) 6) 7) 8) If the thermocycler is off, press the power button to switch on the instrument. Wait for the instrument to boot and display the Power status light. Switch on the computer connected to the thermocycler. Ensure that the plate has been centrifuged for 1 min at 1000 g to remove any bubbles. Open the tray and place the plate in the precision plate holder with the last row (row H) facing front. Make sure the plate is properly aligned in the holder, well A1 should be positioned at the top-left corner of the tray. To close the tray door, press the tray to move it into the instrument while applying pressure to the right side of the tray at an angle. Open the ABI 7500 v2.0.4 software. Select File  New Experiment  From Template. Select the qBiomarker_Mutation_PCR_Array_Template file and click Open. Very that run method is correct. Select Run Menu. Click Start Run in Run Status window. After the PCR Run Page 3 of 4 QIAGEN GmbH  QIAGEN Str. 1  40724 Hilden  Germany  Commercial Register Düsseldorf (HRB 45822) Managing Directors: Peer M. Schatz, Roland Sackers, Bernd Uder, Dr. Joachim Schorr, Douglas Liu
  • 4. Technical Note 1) 2) To determine CT values, Set Baseline at 3 to 15 cycles and Threshold at 0.1. Export CT values. Page 4 of 4 QIAGEN GmbH  QIAGEN Str. 1  40724 Hilden  Germany  Commercial Register Düsseldorf (HRB 45822) Managing Directors: Peer M. Schatz, Roland Sackers, Bernd Uder, Dr. Joachim Schorr, Douglas Liu

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