UVP HybriCycler - Manual (Page 10)

HC-3000 HybriCycler Hybridization Oven 10
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 target
mismatching Lower salt and temperature
Target and probe is
degraded on aqueous
solution Hybridize in a formamide-based buffer
Unacceptable high
background
Use less probe
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
4: Aqueous or Denaturing Hybridization Buffer
If hybridization takes place in an aqueous salt environment of 0.8 to 1.2M salt,
the T
M½
(the temperature at which the half of the duplex molecules will dissociate
under a given set of conditions) can be 90 °C. This is high enough to degrade
DNA, RNA and some proteins. It is therefore possible to add formamide as a
denaturing / temperature lowering agent because for every percent of formamide
in the reaction the T
M½
is reduced by 0.65 °C. Therefore, at 80% formamide,
reactions can be performed in the 40 - 55 °C range. However the rate of
formamide-based hybridization is at least three-fold lower than that of aqueous
hybridization requiring longer incubations.