Since  LoTemp™ PCR is a new approach for cycle primer extension, the users may have some general questions to which we are providing the following answers for better understanding of this technology. 

Q. How do you define LoTemp™?

 A. In repeated cycle DNA primer extension, LoTemp™ refers to a lowered denaturing temperature in the range of 70-85ºC compared to the traditional PCR which depends on heating the reaction mixture to 94-95ºC for template denaturing.

 Q. I have no experience in denaturing double-stranded DNA (dsDNA) at 85ºC. How can it be possible?

 A. It is well known that dsDNA can split into two complementary single-stranded DNA molecules below 95ºC in the presence of a variety of melting agents. LoTemp™ HiFi® ready-to-use PCR mix contains an appropriate mixture of melting agents that force the denaturing  of dsDNA templates at 85ºC, the upper limit of heat that HiFi® DNA polymerases can survive during thermocycling.

 Q. Can I reduce the denaturing temperature further to below 85ºC in performing LoTemp™ PCR? 

A. Yes. But it is not possible when the current LoTemp™ PCR mix is used. The melting agents in the currently available ready-to-use LoTemp™ mix have been optimized for 85ºC denaturing and for enzymatic polymerization of the HiFi® DNA polymerase system already incorporated in the mix.

 Q. Can I raise the denaturing temperature to above 85ºC in performing LoTemp™ PCR? 

A. No. At a temperature above 85ºC, some, although not all, of the HiFi®  DNA polymerases in the LoTemp™ mix will be irreversibly inactivated. 

 Q. Is it possible to leave dNTPs in the working solutions at room temperature for several weeks?

 A. Yes. We have successfully incorporated certain protective reagents in the LoTemp™ HiFi® ready-to-use PCR mix to drastically reduce the rate of dNTP degradation at room temperature.  

Q. What is the definition of room temperature?

A. We define room temperature as 15-25ºC, or 59-77ºF. At this temperature, the LoTemp™ HiFi® ready-to-use PCR mix has proved to be stable for several weeks in field studies.