REAL-TIME PCR continued
Table 1 – Ct values of the samples with and without concentration step (RFU = relative fl uorescence units) Name of sample Negative control
Fluorescence dye FAM
Without concentration Concentration with Vivaspin 20 (incl. coating buff er)
FAM FAM FAM FAM FAM FAM FAM FAM FAM FAM FAM FAM
2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000
Threshold value (RFU) 2000
Ct-value No Ct
No Ct No Ct 24.60 24.87 17.16 17.02 16.72 17.18 16.91 16.25 16.91 16.25
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• Brief mixing of the rehydrated solutions with the vortex mixer and short centrifugation.
• Composition of the master mix: per reaction, pipetting and mixing of 49 μL primer/probe/nucleotide mix (red cap) and 1 μL of the internal control (yellow cap) into a 1.5-mL reaction tube.
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Table 2 – ΔCt values with and without the use of coating buff er Membrane treatment
Sample volume
Vivaspin 20
(VS20)
VS20 + 18 mL sample for 10 min 3500 × g
VS20 + 18 mL sample + 2 mL coating buff er for 20 min 3.500 × g
AMERICAN LABORATORY • 10 • JUNE/JULY 2014 18 mL ΔCt 6.07 ®
• Centrifugation of the reaction tubes contained in the kit with the lyophilized components for 5 sec at max. speed. Addition of 1275 μL rehydration buff er to the primer/probe/nucleotide mix (red cap).
• Addition of 300 μL water (PCR quality) to the positive control (green cap) and to the internal control (yellow cap).
• Five minutes of incubation at room temperature until rehydration was complete.
18 mL
7.90
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