MJ Research DNA Engine Dyad PTC-220, Руководство по эксплуатации

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DNA Engine Dyad Operations Manual
5-12
Choosing a Temperature Hold Time
Because of the calculated melting temperature (Tm) of a DNA hybrid, DNA polymerase
processivity, and reaction kinetics, it may be possible to generalize conditions regarding
thermal-cycling protocols. However, decisions on denaturation, annealing or extension hold
times will be reaction specific and should be optimized.
A target temperature can be held for as little as 1 second, or up to forever, should a protocol
require an extended incubation period. In graphical programs, the maximum programmable
hold time for a step is 1 hour 39 minutes and 59 seconds (99:59), with the exception of a
forever incubation. In advanced programs, the maximum programmable hold time is 18
hours, with the exception of a forever incubation.
Choosing A Thermal Gradient
Molecular biology labs routinely optimize annealing and denaturing temperatures for ther-
mal cycling reactions. Optimization is critical, but not always easy. The Tm (‘melting tem-
perature’) of an oligonucleotide can be estimated using an empirically derived correlation
which considers a combination of DNA length, G+C content, and salt concentration. How-
ever, since the Tm is only an estimate, the “true” annealing temperature may need adjusting
in the actual experiment. This optimization involves repeating a reaction at several different
annealing temperatures, which requires a great deal of time and monopolizes the instrument
while several experiments are run in tandem. To complicate matters further, similar time-
consuming experiments may also be required for denaturing temperature optimization.
The Dyad cycler programmable temperature gradient feature allows for optimization of an
incubation temperature in a single experiment by analyzing a number of different tempera-
tures simultaneously. The thermal gradient delivers a controlled thermal difference, left to
right, across the sample block. This will result in a precisely defined temperature gradient that
is repeatable from experiment to experiment. The range of temperatures that can be achieved
from left to right across a 96-well Alpha unit can be as small as 1
°
C or as great as 24
°
C. The
maximum programmable temperature is 105
°
C; the minimum programmable temperature is
30
°
C.
Note: The programmable temperature gradient feature is only accessible if a 96-well Al-
pha™ unit(s) is mounted in the Dyad cycler. The gradient feature is not compatible with other
types of Alpha units.
The temperature of any well or column in the sample block may be displayed using the
<Gradient Calculator> available from the <Tools> drop-down menu in the Status window.
Since our oligonucleotide annealing temperature is not optimized, we will replace our an-
nealing step with a gradient step. We will optimize in the range of 45
°
C to 65
°
C.