UVP HybriCycler - Manual (Page 9)

HC-3000 HybriCycler Hybridization Oven 9
1: Concentration of Species
Target: How much target molecule depends on the species you expect to find.
Cellular constituents may be expressed in large or small amounts; the trick is to
start with enough target (~25
µg) and determine experimentally.
Probe: plan to have more probe than expected target. To answer questions
about adding too much probe; run an extinction experiment: serially increase the
amount of target by a factor of two and use a fixed amount of probe. Hybridize
for a short length of time and quantitate the amount of probe that has hybridized.
As long as the signal increases and shows linearity there is excess probe (Fig 2
left). If the signal levels off and a loss of linearity noticed, then the probe is not in
excess (Fig 2 right).
2: Length of Probe
The goal is to increase hybridization efficiency while minimizing background. In
most cases probes range from 20 – 1000 bps.
3: Salt Concentration and Temperature
Nucleic acid requires salt (monovalent cations) to reduce the ionic effects of the
phosphate backbone, and heat as a form of non-denaturing kinetic energy.
Because the salt concentration and temperature effect each other, knowing the
thermostability of the hybrid probe is helpful. Hybridization rate varies directly
with the sodium ion concentration between 0.03 and 1.2 M. Most protocols run
between 0.5 and 1.1 M Sodium.