Creating Programs
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Beyond the Example Protocol: Other
Considerations
In addition to the above considerations, you can also include other protocol varia-
tions which will further optimize the yield and quality of your product.
For example, an initial extended denaturation step can serve to destroy any heat-
labile nucleases and other potentially interfering components, while ensuring that the
nucleic acid has been completely denatured and prepped for annealing.
In some protocols, after the final elongation step, a slow temperature ramp can also
be included to ensure proper product annealing.
In addition, some protocols can include a sustained incubation at sub-ambient tem-
peratures to preserve the integrity of the products.
We will choose an initial incubation at 94°C for 1 minute before cycling, and a final
incubation of the sample at 10°C forever. Your written program might now appear as
follows:
Raw program:
Use calculated temperature control mode
Use constant lid control mode at 100°C
An initial incubation at 94°C for 1 minute
1.
92°C for 30 seconds
2.
Gradient from 45°C to 65°C for 3 minutes
3.
Goto step 1, 29 more times
4.
An incubation at 10°C forever
5.
END
Now that we’ve made some important decisions regarding the implementation of our
program, we are ready to begin entering steps.
