Improvement | Effect |
Volume of sample | 4 ml |
Volume of IC DNA | 4 ml (0.1 ml concentrated IC discarded at first, 0.2 ml IC in 3.8 ml sterile H2O) |
SYBR Green I | Diluted 1:20 in Dimethyl sulfoxide (DMSO). Volume equals to dead space in Pump B (20 μL) |
Incubation of sample | 13 min at 60˚C |
Incubation of IC | Less than one minute at ambient temperature |
Flow rate and time | 3.5 μL・s−1 of 180 sec and 10 sec |
Measurement wavelength interval | 520 - 530 nm |
Fluorescence count | Average of readings every one sec for 180 and 10 sec |
Background reading (auto-fluorescence) | To be subtracted from sample reading. Fluorescence reading of sample without dye, for correction of any auto-fluorescence of the sample water. Background reading is not subtracted from internal control readings. |
Cleaning | Sterile H2O and air between sample and IC measurement. Acid, alkaline, sterile water and air at the end of a cycle. No air through the flow cell. |
Time consumption | 75 min for one full cycle, including washing/cleaning |