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
