RIDA GENE real-time PCR DNA Assays

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Mervin Lawrence Wade
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1 R-Biopharm AG RIDA GENE real-time PCR DNA Assays Set-up for ABI7500/FAST (Software Version.0) R-Biopharm for reliable diagnostics.

2 Step : To set up a template for DNA Assays After opening the ABI7500 software, click on the New Experiment Tab to set-up the required detection format template for the RIDA GENE real-time PCR DNA assays. page 0/05

3 Step : The following window opens page 3 0/05

Step 3: In the Comments (Optional) field, enter RIDA GENE xxxx and Lot #. 3 Dependent on the instrument, choose either 7500 (96 wells) or 7500 Fast (96 wells) from the instrument selection.

4 Step 3: In the Comments (Optional) field, enter RIDA GENE xxxx and Lot #. 3 Dependent on the instrument, choose either 7500 (96 wells) or 7500 Fast (96 wells) from the instrument selection Choose Quantitation Standard Curve from the experiment type selection. Choose Other from the reagents selection. 5 In the presence of a 7500 Fast instrument, select Fast for the ramp speed, otherwise select Standard. 5 page 4 0/05

5 Step 4: Navigate to the Plate Setup Tab on the left hand side of the screen. page 5 0/05

6 Step 5: Go to the Define Targets and Samples Tab. The respective reporter channels can be defined from the drop down menu. 3 NOTE: For all Targets, select None in the Quencher field from the drop-down menu. 3 page 6 0/05

7 Step 6: To add further targets, click on Add new Target for each respective new target. 3 Select the respective reporter channels in the Reporter field for following dyes: FAM, VIC, ROX, Cy5. NOTE: For all Targets, select None in the Quencher field from the drop-down menu. 3 In the Define Samples field, the NTC can be also safed in the template. 4 Select Add new sample to add another Sample name field. In the Define Samples field, the PTC can be also safed in the template (after Add new sample ) 4 page 7 0/05

8 Step 7: In the Assign Targets and Samples Tab, select None for the passive reference. page 8 0/05

9 Step 8: In the Assign Targets and Samples Tab one can deposit one sample well which contains all previously programmed target channels. Highlight well H in the View Plate Layout Tab. page 9 0/05

10 Step 9: Assign all previously programmed targets to the highlighted well by ticking the box in the Assigns field for all targets. The ticked targets will all be deposited in well H. page 0 0/05

Step 0: Navigate to the Run Method tab on the left hand side of the

4 Stage type Temperature [ C] Time [mm:ss] Ramp rate Data collection

11 Step 0: Navigate to the Run Method tab on the left hand side of the screen. Enter the following run protocol according to the RIDA GENE insert of a DNA Assay. 4 Stage type Temperature [ C] Time [mm:ss] Ramp rate Data collection Holding stage 95 0:00 00 % off 3 Cycling stage 45 x Holding stage 95 00:5 00 % off 60 00:30 00 % on 40 00:0 00 % off 3 Optionally, a cooling step can be included. 4 The reaction volume has to be set to 5. page 0/05

12 Step : To save this template, click on File. Save as Template.. page 0/05

13 Step : A new window opens: Name the file RIDAGENE DNA. Safe as *.edt file (b) in your respective templates folder. page 3 0/05

14 Step 3: To start a run After opening the ABI7500 software, click on the drop down menu of the New Experiment Tab. Select From Template. page 4 0/05

15 Step 4: A new window opens: Select the previously programmed template RIDAGENE DNA from your respective template folder Open the template. page 5 0/05

16 Step 5: A new window opens: In the Comments (Optional) field, enter the respective assay and the Lot # of the Assay. page 6 0/05

17 6. Step: Navigate to the Plate Setup Tab on the left hand side of the screen. page 7 0/05

18 7. Step: In the Define Targets and Samples Tab, the target names of the respective assay to be run can be defined. For example as shown here, for RIDA GENE MRSA: SCCmec/orfX, ICD, S. aureus, meca/mecc. page 8 0/05

19 8. Step: In the Define Samples Tab, the respective sample name can be typed in. To add further samples, click on Add New Sample and type in the respective sample names of all samples. page 9 0/05

20 9. Step: Navigate to the Assign Targets and Samples Tab and the View Plate Layout Tab. 3 Highlight the saved sample template in well H and right click on it with your mouse. 3 page 0 0/05

21 0. Step: A new window opens: Select copy from the given options. page 0/05

NTC and PTC. Right click on the wells with your mouse.

22 . Step: With the cursor, highlight the respective wells that need to be measured according to your sample size, including NTC and PTC. Right click on the wells with your mouse. A new window opens: Select Paste from the given options. page 0/05

23 . Step: All selected wells will now have all targets enabled. Now, define your individual samples and controls on your plate by doing the following: Highlight the respective well for the NTC, for example Well A. 3 Enable NTC by ticking the respective Assigns box. The NTC has now been assigned to Well A in this particular example. 3 page 3 0/05

24 3. Step: To define the PTC on the plate and all samples, repeat step for each well required: Highlight the respective well for the PTC, for example Well A. Enable PTC by ticking the respective Assigns box. The PTC has now been assigned to Well A in this particular example. page 4 0/05

25 4. Step: To define the individual samples on the plate, repeat step for each well required: Highlight the respective well for the samples, for example Well B. Enable Sample by ticking the respective Assigns box. Sample has now been assigned to Well B in this particular example. page 5 0/05

26 5. Step: To clear the template well (H) highlight the well. Right click and select clear from the new window. page 6 0/05

27 6. Step: To start the run, press the Start Run Button in the upper right corner. NOTE: A new window will open. The run needs to be saved first before the instrument will start the run. page 7 0/05

28 R-Biopharm AG R-Biopharm Contacts: Information: Phone: +49 (0) Fax: +49 (0) mdx@r-biopharm.de Orders: Phone: +49 (0) Fax: +49 (0) orders@r-biopharm.de 0/05

HID Real-Time PCR Analysis Software

USER GUIDE HID Real-Time PCR Analysis Software Version 1.3 for use with: 7500 Real-Time PCR Instruments QuantStudio 5 Real-Time PCR Instruments (with 0.2-mL 96-Well Block) Quantifiler DNA Quantification