SYBR™ Green PCR Master Mix

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Amplicon size should be consistent for each target sequence and limited to approximately 65 - 100 bp	PCR plates, tubes and pipette tips should be UV sterilized for 20-30 mins

Upstream tips
Amplicon size should be consistent for each target sequence and limited to approximately 65 - 100 bp
Protocol tips
PCR plates, tubes and pipette tips should be UV sterilized for 20-30 mins

Publication protocol

For the quantitative analysis, plasmids containing the target genes were used as standard. PCR amplification was initially performed for the 16S rRNA of P. endodontalis, P. gingivalis and T. forsythia. The amplicons were cloned using the TOPO TA Cloning Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions and plasmids were transformed into E. coli. The dilutions were used as template DNA in the qPCR reactions. In each reaction, data obtained from the standard curve were used to convert the CT scores (cycle threshold) obtained with patient samples into the exact numbers of DNA copies19,20. The detection and quantification by qPCR was performed using universal (Applied Biosystems®)21 and specific primers for P. gingivalis (forward: 5’-ACC TTA CCC GGG ATT GAA ATG-3’, reverse: 5’-CAA CCA TGC AGC ACCT AC ATA GAA-3’- 83pb), T. forsythia (forward: 5’-AGC GAT GGT AGC AAT ACC TGT C-3’, reverse: 5’-TTC GCC GGG TTA TCC CTC-3’- 88pb) and P. endodontalis (forward: 5’-GCT GCA GCT CAA CTG TAG TCT TG-3’, reverse: 5’-TCA GTG TCA GAC GGA GCC TAG TAC-3’-110pb) (Applied Biosystems®)22 .The species-specific primer sets were designed based on the variable regions of each target gene. The specificity of the primers was confirmed by multiple alignments of relevant sequences from closely related species and by a Basic Local Alignment Search Tool (BLAST) homology search16. The qPCR reactions were performed with the use of a Step OneTM qPCR System (Applied Biosystems®). All reactions were performed in duplicate, and average values were used to calculate the bacterial load. The total volume of each reaction was 10 μl containing 5 μL of SYBR Green ER qPCR SuperMix Universal (Invitrogen Tech-LineSM), 0.1 μM of each primer pair (Applied Biosystems®) and 50 ng/µl template DNA. The thermocycling program included incubation at 95°C for 10 minutes, 40 cycles of 95° C for 15 seconds and 60°C for 1 minute. After the PCR reactions, the dissociation curve (melting curve) was obtained using temperatures between 60° C and 95° C to determine primer specificity. Melting curve analysis revealed only one peak of amplification. All reactions were performed in 48-well MicroAmp optical plates covered with optical adhesive (Applied Biosystems). Data were analyzed by Step OneTM software (Applied Biosystems).

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Manufacturer protocol

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