Hoefer HB1000 - Instrukcja (Strona 23)

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2. Length of probe
The goal is to increase hybridization efficiency while
minimizing background. The larger the probe, the
more efficiently it binds target. The smaller the probe,
however, an ever greater number of probe molecules
is present per unit amount (ng). In most cases probes
range from 20 – 1000 bps.
3. Salt concentration and temperature
Nucleic acid hybridization requires both salt (monova-
lent cations, such as Na
+ ), to counter the charge
effects of the phosphate backbone, and heat, to
provide non-denaturing kinetic energy that drives the
slow nucleation step. Because the effects of each
variable affect the other, knowing the thermostability
of the hybrid probe-target is helpful. The hybridization
rate varies directly with the sodium ion concentration
between 0.03 and 1.2 M. Most protocols call for an
environment between 0.5 and 1.1 M Na
+ .
Situation Response
G+C = 45 – 55% Follow normal protocol.
G+C < 45% Lower salt and temperature.
G+C > 55% Raise salt and temperature.
Evidence of probe Lower salt and temperature.
target mismatching.
Target and probe
Hybridize in a
are degraded in an formamide-based buffer
aqueous environment. solution.
Unacceptable high Use less probe.
background.
Hybridize at lower salt /
higher temperature.
Wash with lower salt / higher
temperature.
Incubate with very low salt;
change nuclease (solution).
Use a smaller probe or a
different probe.
Clean probe of contaminants
prior to use.