Save
Nucleic Acid Hybridization
Hybridization Conditions
Save
Share
Learn
Content
Leaderboard
Learn
Created by
Justine Isabella
Visit profile
Cards (18)
Degree of homology between the Probe and the Nucleic Acid.
Stringency
If the Stringency is high;
More demanding
in terms of
complementarity.
If the Stringency is low;
Less
demanding in terms of
complementarity.
If the Stringency is too high;
No binding
will occur
If the stringency is too low;
Nonspecific Binding
Factors affecting Stringency
Temperature
Salt
Concentration
Denaturant
Concentration
Length
and
Nature
of
probe
Stringency is HIGH when there is?
High
Temperature
Low
Salt
Concentration
High
Denaturation
Concentration
Stringency is LOW when there is?
Low Temperature
High Salt Concentration
Low Denaturant Concentration
Formula for Temperature
Tm=
81.5
degrees celcius +
16.6
logM +
0.41
(%GC) -
0.61
% (% formamide) - (
600
/
n
)
It is expressed as Cot and is the length of unique (nonrepetitive) nucleotide sequences.
Sequence Complexity
Formula for Sequence
Complexity
Cot
= [
initial Dna
] x
time
for
reannealing
Sequence Complexity: More
complex
= more
time
to
reassociate
Temperature
25
degrees
celcius
below
Tm for 1-3 Cot 1/2 is optimal for
dsDna
probes.
It is the limiting agent during hubridization in the incubation system.
Probe
Incumabtion System: Sensitivity Increases with?
Probe Concentration
Incubation System: Keep volume of hybridization solution?
Low
Incubation System: Recommended hybridization buffer volume ______ of the membrane surface are.
10
mL/
100
cm^2
Incubation within a sealed bag in a water bath or
capped
glass
cylinders in rotary ovens maintains
optimal
hybridization
temperature.