BIO RAD CFX384, Instruction Manual

Page: 48 / 158

CFX96 and CFX384 Systems Manual
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For example, a typical PCR protocol includes the following three sets of steps with a total run
time of 1.5 to 2.0 hours:
1. Initial template denaturation and enzyme activation (95°C for 3-10 minutes).
2. Cycles of three temperature steps (30 to 40 cycles): Denaturation of template (94-95°C
for 15-30 seconds), annealing of primers (anneal for 15-30 seconds), and extension of
product (72°C for 15-60 seconds).
3. Final extension (72°C for 10 minutes).
The Protocol AutoWriter might make these modifications to shorten a typical protocol:
• Change the initial template denaturation and enzyme activation step from 95°C for 3
minutes to 98°C for 30 seconds
• Change the denaturation step in each cycle from 95°C for 30 seconds to 92°C for 1
second
• Combine the annealing and extension steps into a single step at 70°C for 20
seconds
NOTE: Combining the annealing and extension steps imposes limits on the melting
temperature of the primers. If the melting temperatures of the primers do not fall
within the specified range, adjust the primers. For example, shorten the primers by
2 to 3 basepairs (bp), or redesign them to adjust the melting temperature.
Create a Protocol With the Protocol AutoWriter
Follow these steps to use the Protocol AutoWriter to create a new protocol:
1. Click the Protocol AutoWriter button on the toolbar to open the Protocol AutoWriter
window.
2. Enter the Annealing Temperature (Ta) and Amplicon Length in the boxes within the
Enter Target Values/Enzymes pane. If you do not know the annealing temperature for
primers, click the
Ta Calculator button to enter the primer sequences and calculate the
annealing temperature. For information about the calculations used in the Ta Calculator
see Breslauer et al. 1986.
3. Select an enzyme type from the list of options (iTaq, iProof, or Other).
4. Add parameters in the Additional Parameters (Optional) pane if you want to add a
Gradient Range, Hot Start Activation temperature, or Final Extension time in the
protocol.
5. Select a protocol speed (Standard, Fast, or Ultrafast) by moving the sliding bar in the
Type pane. When you move the sliding bar, the software adjusts the total run time. Select
Real-time PCR to tell the software to collect fluorescence data.
6. Review the protocol in the Preview pane and total run time. Make changes as needed.
TIP: Enter the lid temperature and sample volume before each run by editing the
parameters in the Start Run tab (see “Start Run Tab” on page 24).
7. Click OK to save the new protocol, or click Cancel to close the window without saving
the protocol.
TIP: To edit a protocol written with the Protocol AutoWriter, open the protocol file
(.prcl extension) in the Protocol Editor window (page 31).
NOTE: Bio-Rad Laboratories does not guarantee that running a protocol written in
the Protocol AutoWriter window will always result in a PCR product.