Hoefer HB1000 - Instrukcja (Strona 22)

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p13
Experimental protocols
There are many different protocols available on
the web, in journals, and in textbooks. We refer-
ence several at the end of this section.
1. Concentration of species
Target: The ideal quantity of target molecule depends
on the complexity of the target nucleic acid. The
amplified DNA, for example, may have been expressed
in large or small amounts. Therefore, the trick is to
start with sufficient target (~25 μg) and determine an
ideal amount experimentally.
Probe: Plan to have more probe than expected target.
To determine this, perform an extinction experiment:
serially increase the amount of target by 2-fold against
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 present (Fig 6 left). If
the signal levels off, then the probe is not present in
excess (Fig 6 right).
Fig 6. Analysis for probe excess in
hybridization reactions.