Thermal Cycler Construction
rfwhittier at hotmail.com
Wed Sep 4 20:17:04 EST 2002
Ashwin Nagarajan wrote:
>I am trying to build a thermal cycler for a Polymerase Chain Reaction
>(PCR). Could someone please tell me what the bare minimum rate of cooling
>is that can be used in the cycling process without adversely affecting the
>fidelity of the process? What is the recommended rate of cooling?
Most users could live with a cooling rate of 1oC/s, but asking about
a "minimum rate . . . without adversely affecting fidelity" seems to be
barking up the wrong tree. Minimizing the time of incubation at or near
denaturation temperatures is most important for preserving polymerase
activity. Beyond this, the length of time and temperature setting for
denaturation will affect amplification efficiency and fidelity through
processes such as depurination and deamination of cytosine to uracil.
The briefer the better, as long as the DNA fully denatures. To achieve
this, the accuracy of temperature sensing and uniformity of the heating
block are probably more important. Anyway, even the most meticulous
PCR reagents and conditions still come up short by 1000-10,000 fold
fidelity-wise in comparison to in vivo replication in E. coli, and we
take this into account whenever we make use of PCR.
If you're a high school biology teacher trying to save money, the best
option would be to set up water baths at the desired temperatures,
insert the tubes into floats, and manually transfer the floats between
baths. If you're trying to design a commercial cycler, you'll need much
more precise advice than you'll get in this forum.
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