I want to know how can we make sure that after heating staple and ds DNA, how can I make sure that the ss DNA hybridizes with ss staple and not to their own complementary strand again (i.e ss DNA to ss DNA and ss staple to ss staple).
The situation you describe is the same as in a regular PCR when you use complementary primers. The short fragments hybridize always faster then the longer ones as long as they are complementary and you cant do much about it.. You can try to shift the process on the favour of binding longer fragments with shorter ones only by keeping the renaturation temperature above the melting temperature of the smaller fragments. e.g. the melting temp. for your probe is 55°C then after after heating the sample you cant go lower then this temperature for a while. It will prevent the sample fragments on binding to each other and favour binding to ssDNA. Also try to dilute the mixture, so that the probability of each fragment meeting one another is the same. And even then you will have less amounts of hybrids then normal ssDNA-ssDNA or ssSample-ssSample joins
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