Back to protocols page
The major hurdle to embarking on an AmpliCot experiment is obtaining access to a real time PCR machine. Here are a few pointers:
- If it's possible for you to choose which model of real time PCR machine you use, we would recommend getting an MJ Opticon (Bio-Rad). These machines have a robust design, a convenient interface, and a gradient feature. The gradient feature is convenient for automated selective denaturation of the sample wells (while the reference wells are left unmelted) but there are other ways of selectively denaturing the sample wells--it can be done manually, or the samples and references may be run on different machines or at different times. If you have an Opticon 1, with version 1 of the Opticon Monitor software, you should be able to use the machine programs and software on this website without modification; two-color machines may require slight modifications.
- In principle any real time machine can be used as long as its software permits long holds with repeated fluorescence reads during the annealing reaction. The software for some ABI models (e.g. ABI7500) is not flexible enough for this application.
- If you are borrowing time on a machine with many users, the initial amplification steps of the protocol may be performed as regular PCR reactions, rather than using real-time PCR. (Real-time PCR is helpful for quality assurance and trouble shooting but is not absolutely necessary.) The annealing steps are easily done overnight, leaving the machine free for other users during the day.
Other useful information is in our faq.
Last updated September 5, 2006