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Repetitive reaction for random priming?

Repetitive reaction for random priming? - Molecular Biology Techniques

Repetitive reaction for random priming? - Molecular Biology Forum. Includes forums for common molecular biology techniques.


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Old 06-07-2009, 02:41 AM
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Smile Repetitive reaction for random priming?



Dear all,

I am a PhD student who starts doing random priming to label hot probes recently. I have a question.
I wonder whether there is a way to repeat reaction (like 30 cycles) to make sure more random primers are used to synthesize more hot probes?
Since my protocol (adapted from Molecular Cloning 3rd edition) incubates the reaction mixture for just one cycle (1 hour) at room temperature, I suppose there should be left many random primers which were not used in the reaction. As romdom primers can be bound to any sequences including newly synthesized hot probes, I wonder the amount of labgels probes would increased exponentially if the reaction can be done with repetitive cycles such as PCR.
One problem is that Klenow fragment which I use for the reaction would be heat inactivated durinf DNA denaturation......


Because I am novice to the field, I might have critical mistake in the rationale above.
I would like to have comments and opinions from everyone.

Thank you.
Rio Sugimura
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hybridisation , priming , random , reaction , repetitive


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