In article <54513t$m4f at cardinal2.Stanford.EDU>, ladasky at leland.Stanford.EDU
says...
>>Greetings, everyone,
>stuff deleted
>> I think that the problem may be with the cycling program. Anyone
>who works with the Robocycler should know that one does not enter the time
>at which a sample sits at a temperature -- rather, one enters the total
>time that a sample sits on the block, including ramp time. I was told that
>the rule of thumb is to assume a ramp rate of one second per degree Celsius.
>Thus if I want the sample to incubate for 30 seconds at 95 degrees following
>a 72-degree extension, I would enter a time of 53 seconds (23 seconds to
>ramp plus the 30 second "hold").
>> This formula seems too simplistic to me. Could I be overexposing my
>DNA to destructive high temperatures? Is there a better rule of thumb for
>calculating the time on the block? Do sample volumes matter? Are there any
>other problems I may have overlooked? Please reply if you have any ideas.
>Thanks a lot!
>One degre per second is the value given by Stratagene if you call them but,
like you, I find it to simple and in fact very slow. I suspect they are
extremely far on the safe side with this value. Did anybody try to actually
mesure the rate of heating in this machine with, for instance a small
thermoprobe in one of the tube?
That would be helpfull.
Pascal_Bochet at brown.edu
--
>Unique ID : Ladasky, John Joseph Jr.
>Title : BA Biochemistry, U.C. Berkeley, 1989 (Ph.D. perhaps 1998???)
>Location : Stanford University, Dept. of Structural Biology, Fairchild
D-105
>Keywords : immunology, music, running, Green
