4.3 HYBRIDIZATION CONDITIONS, STRINGENCY Flashcards
What factors affect the stringency in hybridization reactions?
Temperature, salt concentration, denaturant concentration (e.g., formamide)
What term describes the combination of conditions affecting probe and target complementarity?
Stringency
How much does the Tm increase for RNA hybrids compared to DNA hybrids?
20°C to 25°C
What value represents the time required for half of a double-stranded sequence to anneal?
Cot 1/2
hat is the effect of a 1% difference in sequence complementarity on Tm?
Decreases Tm by 1.5°C
What is the simpler formula to estimate Tm for short probes (14-20 bases)?
Tm = (number of GC pairs × 4°C) + (number of AT pairs × 2°C)
What is the hybridization temperature relative to the melting temperature?
5°C below the melting temperature
What term refers to the empirical value used to optimize stringency conditions for Southern or Northern blot analysis?
Tm and Cot values
How much lower should the hybridization temperature be for optimal stringency with double-stranded DNA probes?
25°C below the Tm
What term describes the time required for half of a sequence to reanneal under specific conditions?
Cot 1/2
What is the recommended hybridization buffer volume per 100 cm² of membrane surface area?
10 mL
Which chemical in the hybridization buffer lowers the optimal hybridization temperature, especially useful for RNA probes?
Formamide
What is a typical hybridization time for short probes (less than 20 bases)?
1 to 2 hours
How long does hybridization usually take for probes longer than 1,000 bases in Southern or Northern blots?
16 hours or more
What inert polymers can accelerate hybridization rates for probes longer than 250 bases?
Dextran sulfate, polyethylene glycol, polyacrylic acid
