Protocols: Real Time PCR


AB 7300 Real Time PCR System
AB 7900 HT Fast Real Time PCR System
Cepheid SmartCycler

AB 7300 Real Time PCR System

AQ Protocol

AB 7900HT Fast Real Time PCR System

AQ Protocol | RQ Protocol

Cepheid SmartCycler

1. Spin down your samples.
2. Double-click the Smart Cycler icon on the desktop, or for the windows Start menu select Programs -> Cepheid -> Cepheid Smart Cycler.
3. The splash screen should appear, and two "A's" should light up and flash on the top of the Smart Cycler processing block.

Step 4 to 8 is necessary if you don't have a predefined protocol on the machine. The numbers used here are for your reference. You should use your corresponding values that work best with your experiments.

4. Click the Define Protocols icon to define your protocol.
5. Click the New Protocol button to open the New Protocol dialog box. Enter your protocol name and click OK.
6. Define Stage 1:
a. Enter 95 in Temp column.
b. Enter 150 in Secs column.
7. Define Stage 2:
a. Select 2-Temperature Cycle from the drop-down menu.
b. Enter 40 in the Repeat field at the top of the Stage 2 box to specify that it should be repeated 40 cycles.
c. Enter 95 for Temp and 15 for Secs in the first step.
d. Enter 68 for Temp 30 for Secs in the second step.
e. Click in the Optics cell of the second step and select On from the drop-down menu. This sets detection of the fluorescence signals to occur at the end of the second step in each cycle.
8. Click the Save Protocol button.

9. Click the Create Run icon to create a new run. Enter your run name in the Run Name field.
10. Click the arrow in the Dye set box and select FTTR25 for 25 μl Smart Cycler reaction tubes or FTTR 100 for 100 μl reaction tubes.
11. Click the Add/Remove Sites button to open the Select Protocols and Sites dialog box. Select your protocol and sites for the reactions.
12. Review the run setup and click Start Run. (The run normally takes 1 hr 15 minutes.) To do data analysis, do the following:
13. Select Results Table in Views. Set up the Results Table table as follows for each site:
14. Select STD from Sample Type drop-down menu for each standard.
15. Enter the concentration of each standard.
16. Select Standard in Views.
a. The Standard graph plots standards (STD) as blue diamonds and unknowns (UNKN) as red rectangles.
17. Click Export to export your results (in spreadsheet format).

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