Stratagene Robocycler, optimizing long PCR

Pascal_Bochet Pascal_Bochet at brown.edu
Thu Oct 17 09:00:10 EST 1996


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




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