Hoefer HB1000 - Instrukcja (Strona 24)

•
p15
4. Aqueous or denaturing
hybridization buffer
If hybridization takes place in an aqueous salt
environment of 0.8 to 1.2 M salt, the T
m½
(the
temperature at which 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 may be necessary to add
formamide as a denaturing / temperature lowering
agent. For every percent of formamide added to 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. On the other hand, the presence of
formamide requires longer incubations, since the rate
of formamide-based hybridization is at least three-fold
lower than that of aqueous hybridization.
Protocol
1
Random priming method for tagging
DNA with fluorescein-labeled nucleotide
or other labeled nucleotides
This method uses DNA polymerase to incorporate
fluorescein-11-dUTP into double-stranded DNA
probes. This protocol can also be used to incorporate
any tagged nucleotides.
Equipment
• Micropipettes and tips
• Microcentrifuge
• 1.5 mL microcentrifuge tubes
• Cap lock for microcentrifuge tube
• Boiling water bath
• Water bath set to 37 °C