Stratagene Robocycler, optimizing long PCR

Gary Truett GaryTruett at POSTOFFICE.PBRC.EDU
Thu Oct 17 09:24:05 EST 1996


Two easy possibilities that you didn't comment on:
1.  Use plasticware from Stratagene.  Their tubes are designed to fit their
heating blocks and other tubes may not perform as well.
2.  Be sure to add mineral oil to block 1 and to cover your reactions with a
small amount of mineral oil.
Both of these suggestions are made in the Robocycler manual.


>Greetings, everyone,
>
>        I have some PCR amplifications I'm working on in the 5 Kb - 7 Kb
>range.  They are proving to be difficult to optimize by temperature alone,
>so I am now going to look at temperature in conjunction with cosolvents,
>magnesium concentration, etc.  For enzymes, I'm using Perkin Elmer Taq
>polymerase and Stratagene's Taq Extender (Pfu polymerase).  A neighboring
>lab has generously offered to let me use their Robocycler 40, which has a
>gradient temerature block.  In theory, this should speed up my work tre-
>mendously.  Unfortunately, the folks who own the Robocycler have *lost the
>manual*, and I'm working on the basis of rules of thumb.
>
>        I have started conservatively, and have met with disaster already.
>One of my lab mates regularly performs a 3.1 Kb PCR that only works with
>Taq Extender, not with Taq alone.  I have successfully duplicated this 
>reaction on our lab's workhorse thermal cycler, a Perkin Elmer 9600.  Then
>I took the exact same reaction mixtures over to the Robocycler.  I got 
>smears instead of bands.  In my experience, this can happen in PCR's when
>DNA is broken by lengthy incubation a high temperatures. giving non-speci-
>fic ends that can anneal and extend and compete with the desired reactions.
>
>        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!
>
>-- 
>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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