The total volume used in this protocol is 50 pl, however if necessary, each reaction can be made in a total volume of 25 pl maintaining the correct concentrations of reagents:
2 x SYBR® Green Master Mix Forward primer (10 ^M) Reverse primer (10 ^M) Molecular biology grade H2O Specimen or control nucleic acid extract Total
Carefully pipette the reaction mixture into wells of MicroAmp® Optical 96-well Reaction Plate (Applied Biosystems). Use 107-102 cell dilutions of P. intermedia to make a standard curve in each individual reaction plate and use 5 ^L of molecular biology grade H2O as negative control. Add 5 ^L DNA in each sample well.
Place the reaction plate into GeneAmp® 5700 Sequence Detection System (PE Applied Biosystems).
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