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Primer specificity is affected by a number of factors, including sequence, primer location, and the RT-PCR system used.

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At the end of the PCR reaction, the specific sequence will be accumulated in billions of copies ().

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Even if our approximations don't give accurate absolute numbers (since we are not using the standard salt concentration or whatever), if they help us design primers with relatively similar annealing temperatures, that is generally what we need.

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The guidelines for primer design vary slightly.

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Applied Biosystems Big Dye Primer Reverse 5000-reaction kit (Cat.

Positive control: This is a control reaction using a known amount of template.

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