Hybridization Conditions

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.